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- | <title>FREiGEM 2009</title> | + | <title>FREiGEM</title> |
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- | <div class="art-Header-png"></div>
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| <ul class="art-menu"> | | <ul class="art-menu"> |
- | <li><a href="https://2009.igem.org/Team:Freiburg_bioware" class="active"><span | + | <li><a href="https://2009.igem.org/Team:Freiburg_bioware"><span |
| class="l"></span><span class="r"></span><span | | class="l"></span><span class="r"></span><span |
| class="t">Home</span></a></li> | | class="t">Home</span></a></li> |
- | <li><a href="#"><span class="l"></span><span | + | <li><a href="https://2009.igem.org/Team:Freiburg_bioware/Team"><span |
- | class="r"></span><span class="t">The Team</span></a>
| + | class="l"></span><span class="r"></span><span |
| + | class="t">The Team</span></a> |
| <ul> | | <ul> |
| <li><a | | <li><a |
| href="https://2009.igem.org/Team:Freiburg_bioware/Team">Overview</a></li> | | href="https://2009.igem.org/Team:Freiburg_bioware/Team">Overview</a></li> |
- | <li><a href="#">Fotos</a></li> | + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Team/Portraits">Portraits</a></li> |
| </ul> | | </ul> |
| </li> | | </li> |
- | <li><a href="#"><span class="l"></span><span | + | <li><a |
- | class="r"></span><span class="t">The | + | href="https://2009.igem.org/Team:Freiburg_bioware/Project"><span |
- | Project</span></a></li>
| + | class="l"></span><span class="r"></span><span |
- | <li><a href="#"><span class="l"></span><span
| + | class="t">The |
- | class="r"></span><span class="t">Parts</span></a>
| + | Project</span></a> |
| <ul> | | <ul> |
- | <li><a href="#">1</a> | + | <li><a |
- | <ul>
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Project#Summary">Summary</a> |
- | <li><a href="#">2</a> </li>
| + | |
- | <li><a href="#">3</a> </li>
| + | |
- | <li><a href="#">4</a> </li>
| + | |
- | </ul>
| + | |
| </li> | | </li> |
- | <li><a href="#">5</a></li> | + | <li><a |
- | <li><a href="#">6</a></li> | + | href="https://2009.igem.org/Team:Freiburg_bioware/Project#Subprojects">Subprojects</a></li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Project#Highlights">Highlights</a></li> |
| </ul> | | </ul> |
| </li> | | </li> |
- | <li><a href="https://2009.igem.org/Team:Freiburg_bioware/Notebook"><span class="l"></span><span | + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Human_Practice"><span |
| + | class="l"></span><span class="r"></span><span |
| + | class="t">Human |
| + | Practice</span></a> |
| + | <ul> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Human_Practice/Ethics">Ethics</a> |
| + | </li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Human_Practice/Safety">Safety</a></li> |
| + | </ul> |
| + | </li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Notebook" |
| + | class="active"><span class="l"></span><span |
| class="r"></span><span class="t">Notebook</span></a></li> | | class="r"></span><span class="t">Notebook</span></a></li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/cloning1"><span |
| + | class="l"></span><span class="r"></span><span |
| + | class="t">Parts</span></a> |
| + | <ul> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/cloning1">Basic |
| + | Parts</a></li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/cloning">Composite |
| + | Parts</a></li> |
| + | </ul> |
| + | </li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Collaboration"><span |
| + | class="l"></span><span class="r"></span><span |
| + | class="t">Collaboration</span></a></li> |
| + | <li><a |
| + | href="https://2009.igem.org/Team:Freiburg_bioware/Modeling"><span |
| + | class="l"></span><span class="r"></span><span |
| + | class="t">Modeling</span></a></li> |
| </ul> | | </ul> |
| </div> | | </div> |
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| <div class="art-Post-inner"> | | <div class="art-Post-inner"> |
| <div class="art-PostMetadataHeader"> | | <div class="art-PostMetadataHeader"> |
- | <h2 class="art-PostHeaderIcon-wrapper">October<span | + | <h2 class="art-PostHeaderIcon-wrapper"> <img |
- | class="art-PostHeader"></span> </h2> | + | style="width: 28px; height: 25px;" alt="" |
| + | src="https://static.igem.org/mediawiki/2009/2/2a/Freiburg09_Post_tanne_2.png" /> |
| + | October<span class="art-PostHeader"></span> </h2> |
| </div> | | </div> |
- | <div style="text-align: center;" class="art-PostContent"> | + | <h3 style="margin-left: 40px;">01.10.09, Hannes, Manu, |
| + | Laura, Gerrit, Sarah, Christoph, |
| + | Caro, Julia, Anika, Isabel</h3> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Tetracycline; 10 x 1ml, Concentration: 25 g/l, |
| + | stored in -20°C, box with antibiotics |
| + | </li> |
| + | <li> Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, |
| + | stored in -20°C, box with antibiotics |
| + | </li> |
| + | <li> plasmid preparation of |
| + | pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 |
| + | </li> |
| + | <li> plasmid preparation of |
| + | pEX-Strep-Dig-Split-Fok(active) Klon 1 |
| + | </li> |
| + | <li> glycerin stock of |
| + | pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 |
| + | (RV308), stored at -80°C |
| + | </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>protein purification of HisFluASplitFoki (expressed in |
| + | BL21de3) with NiNTA column |
| + | </li> |
| + | <li>SDS gel of protein purification HisFluASplitFoki |
| + | [[Image:Freiburg09_011009_pg_HisFluASplitFoki011.jpg|none|thumb|SDS-Gel; |
| + | HisFluASplitFoki; Lanes: NEB prestained protein marker, elution |
| + | fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, |
| + | elution fraction 5, elution fraction 6, elution fraction 7, flow |
| + | through fraction 2, washing fraction 2|400x400px]] |
| + | </li> |
| + | <li>pooled fraction 2-5, dialysis over night in dialysis buffer |
| + | (30mM NaCl, 20mM Tris-HCl, pH 7.4) |
| + | </li> |
| + | <li>phage display: |
| + | - desalt ligation products (for electroporation)</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>pool samples |
| + | (~80µl 445+87/89 and ~52µl 445+87/88)</small><br /> |
| + | <small>add 1 volume of isopropanol, mix</small><br /> |
| + | <small>-80°C for 10 minutes</small><br /> |
| + | <small>centrifuge for 10 minutes at max rpm, 4°C; discard |
| + | supernatant</small><br /> |
| + | <small>add 1 volume of 75% ethanol (without mixing)</small><br /> |
| + | <small>centrifuge for 10 minutes at max rpm, 4°C; discard |
| + | supernatant</small><br /> |
| + | <small>let dry on heat block (50°C), lid open<br /> |
| + | <small>add 25µl water</small><br /> |
| + | <small>thermo shaker for 1h, 45°C, 1000rpm |
| + | - PCR (4 samples each template):</small><br /> |
| + | <small>buffer (with MgCl2): 5µl</small><br /> |
| + | primer #7: 1,5µl<br /> |
| + | primer #1: 1,17µl<br /> |
| + | dNTPs: 2µl<br /> |
| + | Taq: 1µl<br /> |
| + | MnCl2: 0,5µl<br /> |
| + | MgCl2: 5µl<br /> |
| + | water: 33µl<br /> |
| + | DNA (425 or 428): 5µl<br /> |
| + | - PCR:<br /> |
| + | DNA templates: pJs#448 (0,3µl), pJs#449 (0,3µl), |
| + | pJs#375 (1µl), pJs#413 (0,3µl), pJs#445 |
| + | (0,3µl)<br /> |
| + | primers (#95, #7): 1,5µl<br /> |
| + | high fidelity buffer: 5µl<br /> |
| + | dNTPs: 1µl<br /> |
| + | TMenzyme: 0,3µl<br /> |
| + | water: 39,7µl</small><br /> |
| <br /> | | <br /> |
- | <div style="text-align: left;"> | + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | <h3>01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel</h3> | + | <li> Overnight Culture RV308 for Competent Cells, on |
- | <br>* Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
| + | shaker in 37 °C room |
- | <br>* Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
| + | </li> |
- | <br>* plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
| + | <li>analysis of sequences from 28.09.09 |
- | <br>* plasmid preparation of pEX-Strep-Dig-Split-Fok(active) Klon 1
| + | </li> |
- | <br>* glycerin stock of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 (RV308), stored at -80°C
| + | <li>digest of pExStrepDigSplitFoka prep from 30.09.09 |
- | <br>
| + | Plasmid: 15 µl</li> |
- | <br>* protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column
| + | </ul> |
- | <br>* SDS gel of protein purification HisFluASplitFoki
| + | <div style="margin-left: 40px;"><small>water: 9 |
- | | + | µl<br /> |
- | [[Image:Freiburg09_011009_pg_HisFluASplitFoki011.jpg|none|thumb|SDS-Gel; HisFluASplitFoki; Lanes: NEB prestained protein marker, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, flow through fraction 2, washing fraction 2|400x400px]]
| + | BSA: 0.5 µl NEB iGEM stock<br /> |
- | | + | buffer: 3 µl buffer 3 NEB iGEM stock<br /> |
- | <br>* pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4)
| + | Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br /> |
- | <br>*phage display:
| + | Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br /> |
- | - desalt ligation products (for electroporation)<br>
| + | </small><br /> |
- | pool samples (~80µl 445+87/89 and ~52µl 445+87/88)<br>
| + | </div> |
- | add 1 volume of isopropanol, mix<br>
| + | <ul style="margin-left: 40px;"> |
- | -80°C for 10 minutes<br>
| + | <li>digest of PCR product RBSStrepDigSplitFoka from 30.09.09 |
- | centrifuge for 10 minutes at max rpm, 4°C; discard supernatant<br>
| + | Plasmid: 10 µl </li> |
- | add 1 volume of 75% ethanol (without mixing)<br>
| + | </ul> |
- | centrifuge for 10 minutes at max rpm, 4°C; discard supernatant<br>
| + | <div style="margin-left: 40px;"><small>water: 14 |
- | let dry on heat block (50°C), lid open<br>
| + | µl<br /> |
- | add 25µl water<br>
| + | BSA: 0.5 µl NEB iGEM stock<br /> |
- | thermo shaker for 1h, 45°C, 1000rpm
| + | buffer: 3 µl buffer 3 NEB iGEM stock<br /> |
- | | + | Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br /> |
- | - PCR (4 samples each template):<br>
| + | Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br /> |
- | buffer (with MgCl2): 5µl<br>
| + | <br /> |
- | primer #7: 1,5µl<br>
| + | *digest of |
- | primer #1: 1,17µl<br>
| + | pExHisFluaSplitFoki prep from 24.08.09 |
- | dNTPs: 2µl<br>
| + | Plasmid: 15 µl <br /> |
- | Taq: 1µl<br>
| + | water: 9 µl<br /> |
- | MnCl2: 0,5µl<br>
| + | BSA: 0.5 µl NEB iGEM stock<br /> |
- | MgCl2: 5µl<br>
| + | buffer: 3 µl buffer 2 NEB iGEM stock<br /> |
- | water: 33µl<br>
| + | Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br /> |
- | DNA (425 or 428): 5µl<br>
| + | Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br /> |
- | | + | -> put in 37°C room for 2h |
- | - PCR:<br>
| + | ->preparative gel</small><br /> |
- | DNA templates: pJs#448 (0,3µl), pJs#449 (0,3µl), pJs#375 (1µl), pJs#413 (0,3µl), pJs#445 (0,3µl)<br>
| + | <br /> |
- | primers (#95, #7): 1,5µl<br>
| + | </div> |
- | high fidelity buffer: 5µl<br>
| + | <table |
- | dNTPs: 1µl<br>
| + | style="text-align: left; width: 623px; height: 529px; margin-left: 40px;" |
- | TMenzyme: 0,3µl<br>
| + | border="0" cellpadding="0" cellspacing="0"> |
- | water: 39,7µl<br>
| + | <tbody> |
- | <br>* Overnight Culture RV308 for Competent Cells, on shaker in 37 °C room
| + | <tr> |
- | <br>*analysis of sequences from 28.09.09 | + | <td><img alt="" |
- | <br>*digest of pExStrepDigSplitFoka prep from 30.09.09 | + | src="https://static.igem.org/mediawiki/2009/3/3b/Freiburg09_011009_fokverdaue.JPG" /></td> |
- | Plasmid: 15 µl<br> | + | </tr> |
- | water: 9 µl<br> | + | <tr> |
- | BSA: 0.5 µl NEB iGEM stock<br> | + | <td><small>Agarose gel; Lanes: 1.Gene ruler ladder |
- | buffer: 3 µl buffer 3 NEB iGEM stock<br> | + | mix of |
- | Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br> | + | fermentas, |
- | Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br> | + | 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka</small></td> |
- | <br>*digest of PCR product RBSStrepDigSplitFoka from 30.09.09 | + | </tr> |
- | Plasmid: 10 µl <br> | + | </tbody> |
- | water: 14 µl<br> | + | </table> |
- | BSA: 0.5 µl NEB iGEM stock<br> | + | <div style="margin-left: 40px;"><br /> |
- | buffer: 3 µl buffer 3 NEB iGEM stock<br> | + | <small><br /> |
- | Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br> | + | interpretation: |
- | Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br> | + | bands had right size but pExStrepDigSplitFoka showed |
- | <br>*digest of pExHisFluaSplitFoki prep from 24.08.09 | + | unexpectedly low concentration<br /> |
- | Plasmid: 15 µl <br> | + | ->gelextraction <br /> |
- | water: 9 µl<br> | + | ligation of<br /> |
- | BSA: 0.5 µl NEB iGEM stock<br> | + | - pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)<br /> |
- | buffer: 3 µl buffer 2 NEB iGEM stock<br> | + | - pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br /> |
- | Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br> | + | <br /> |
- | Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br> | + | </small></div> |
- | -> put in 37°C room for 2h | + | <ul style="margin-left: 40px;"> |
- | ->preparative gel<br> | + | <small></small><li>Inoculation of - |
- | [[Image:Freiburg09_011009_fokverdaue.JPG|none|thumb|Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka || 400x400px]]
| + | pJS419_StrepDigSplitFoka</li> |
- | interpretation: bands had right size but pExStrepDigSplitFoka showed unexpectedly low concentration<br> | + | <small></small> |
- | ->gelextraction <br> | + | </ul> |
- | ligation of<br> | + | <div style="margin-left: 40px;"><small>- |
- | - pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)<br> | + | pJS419_HisDigSplitFoka<br /> |
- | - pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br> | + | - pEx_HisFluA<br /> |
- | <br>*Inoculation of | + | - pEx_HisDigMiddleLiFoka |
- | - pJS419_StrepDigSplitFoka<br> | + | </small><br /> |
- | - pJS419_HisDigSplitFoka<br> | + | </div> |
- | - pEx_HisFluA<br> | + | <ul style="margin-left: 40px;"> |
- | - pEx_HisDigMiddleLiFoka<br> | + | <li>digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and |
- | <br>*digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning | + | pMA_scFa-anti_NIP for recloning |
- | <br>*Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09 | + | </li> |
- | --> make glycerol stocks tomorrow | + | <li>Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony |
- | <br>*agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning | + | of transformation from 28.09.09 |
- | <br>*cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more) | + | --> make glycerol stocks tomorrow |
- | <br>*gel slices stored at -4°C over night | + | </li> |
- | | + | <li>agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, |
- | <h3>02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max</h3> | + | pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning |
- | <br>* phage display | + | </li> |
- | - OD600 of the pre-culture should be 0,2 <br> | + | <li>cut out insert bands (digestion of pEX didn't work, because |
- | - 37°C shaker<br> | + | there's no EcoRI recognition site any more) |
- | - After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br> | + | </li> |
- | - Apportion culture into 10x50 ml Falkon tubes<br> | + | <li>gel slices stored at -4°C over night |
- | - 15 minutes on ice<br> | + | </li> |
- | - Centrifuge 10 minutes at 4°C / 2500 g<br> | + | </ul> |
- | - discard supernatant, wash pellet with 25 ml H2O (let dry upside down)<br> | + | <h3 style="margin-left: 40px;">02.10.09 Laura, Manu, |
- | - resuspend pellets in 25 ml H2O (10-> 8 tubes)<br> | + | Anika, Julia, Hannes, Gerrit, Isabell, |
- | - 15 minutes on ice<br> | + | Sarah, Caro, Timo, Christoph, Max</h3> |
- | - Centrifuge 10 minutes at 4°C / 2500 g<br> | + | <div style="margin-left: 40px;"><br /> |
- | - Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4 tubes)<br> | + | </div> |
- | - 15 minutes on ice<br> | + | <ul style="margin-left: 40px;"> |
- | - Centrifuge 10 min at 4°C / 2500 <br> | + | <li>phage display |
- | - Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-> 2 tubes)<br> | + | - OD600 of the pre-culture should be 0,2 </li> |
- | - 5 minutes on ice<br> | + | </ul> |
- | - Centrifuge 10 minutes at 4°C / 2500 g<br> | + | <div style="margin-left: 40px;"><small>- |
- | - Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)<br> | + | 37°C shaker<br /> |
- | - 5 minutes on ice<br> | + | - After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br /> |
- | - Centrifuge 10 min at 4°C / 2500 g<br> | + | - Apportion culture into 10x50 ml Falkon tubes<br /> |
- | - Discard supernatant, pool pellets in 1 ml 10% DMSO<br> | + | - 15 minutes on ice<br /> |
- | - Measure OD600 (1:100 dilution), OD should be 0,4<br> | + | - Centrifuge 10 minutes at 4°C / 2500 g<br /> |
- | - Make aliquots à 80 µl, souse with N2<br> | + | - discard supernatant, wash pellet with 25 ml H2O (let dry upside down)<br /> |
- | - Store at -80°C<br> | + | - resuspend pellets in 25 ml H2O (10-> 8 tubes)<br /> |
- | <br>*glycerol stock of pEx_HisFluASplitFoki in BL21de3 | + | - 15 minutes on ice<br /> |
- | <br>*SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09 | + | - Centrifuge 10 minutes at 4°C / 2500 g<br /> |
- | --> do a Western Blot | + | - Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4 |
- | | + | tubes)<br /> |
- | <br>*new ampicilin 100 aliquots | + | - 15 minutes on ice<br /> |
- | <br>*preparation of M13dsDNA | + | - Centrifuge 10 min at 4°C / 2500 <br /> |
- | <br>*chem. competent cells aliqots | + | - Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-> |
- | | + | 2 tubes)<br /> |
- | | + | - 5 minutes on ice<br /> |
- | <br>*Periplasma Project | + | - Centrifuge 10 minutes at 4°C / 2500 g<br /> |
- | | + | - Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)<br /> |
- | Digestion:<br> | + | - 5 minutes on ice<br /> |
- | | + | - Centrifuge 10 min at 4°C / 2500 g<br /> |
- | - digested with xba and spe | + | - Discard supernatant, pool pellets in 1 ml 10% DMSO<br /> |
- | --> GelBilder
| + | - Measure OD600 (1:100 dilution), OD should be 0,4<br /> |
- | | + | - Make aliquots à 80 µl, souse with N2<br /> |
- | <br>* Gelextraction | + | - Store at -80°C</small><br /> |
- | | + | <br /> |
- | Min Elute Gel Extraction Kit<br> | + | </div> |
- | - mesure mass: StrepFokA = 50 mg<be> | + | <ul style="margin-left: 40px;"> |
- | - JS418= 90mg<br> | + | <li>glycerol stock of pEx_HisFluASplitFoki in BL21de3 |
- | -JS419=80mg<br> | + | </li> |
- | - HisFokA = 250mg<br> | + | <li>SDS gel of pool (elution fraction 2-5) from protein |
- | | + | expression (HisFluASplitFoki) from 01.10.09 |
- | Nandropdata<br> | + | --> do a Western Blot |
- | | + | </li> |
- | <br>*Ligation | + | <li>new ampicilin 100 aliquots </li> |
| + | <li>preparation of M13dsDNA </li> |
| + | <li>chem. competent cells aliqots |
| + | </li> |
| + | <li>Periplasma Project |
| + | Digestion:</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- digested |
| + | with xba and |
| + | spe --> GelBilder |
| + | </small><br /> |
| + | </div> |
| + | <div style="margin-left: 80px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Gelextraction |
| + | Min Elute Gel Extraction Kit</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- mesure |
| + | mass: StrepFokA = |
| + | 50 mg<be> |
| + | - JS418= 90mg</be><br /> |
| + | <be>-JS419=80mg</be><br /> |
| + | <be>- HisFokA = 250mg</be><br /> |
| + | <be style="color: rgb(255, 0, 0);">Nandropdata</be></small><br /> |
| + | </div> |
| + | <div style="margin-left: 80px;"> |
| + | <be></be></div> |
| + | <div style="margin-left: 40px;"><be></be><br /> |
| + | <be></be></div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li><be>Ligation |
| - with Quickligase | | - with Quickligase |
- | | + | JS119-StrepFokA</be></li> |
- | JS119-StrepFokA<br> | + | </ul> |
- | JS118-Strep<br> | + | <div style="margin-left: 40px;"><small><be>JS118-Strep</be><br /> |
- | JS118-HisFokA<br> | + | <be>JS118-HisFokA</be><br /> |
- | JS19-HisFokA<br> | + | <be>JS19-HisFokA</be><br /> |
- | | + | <be>- 15 min at 25°C |
- | - 15 min at 25°C | + | </be></small><br /> |
- | | + | </div> |
- | <br>*Transformation | + | <div style="margin-left: 80px;"> |
- | - 2YT-Medium 950 µL to 50µl cells and DNA <br> | + | <be></be></div> |
- | | + | <ul style="margin-left: 40px;"> |
- | BL21-JS190<br> | + | <li><be>Transformation |
- | BL21-JS119StrepFokA<br> | + | - 2YT-Medium 950 µL to 50µl cells and DNA </be></li> |
- | BL21-JS118Strep<br> | + | </ul> |
- | BL21-JS118HisFokA<br> | + | <div style="margin-left: 40px;"><small><be>BL21-JS190</be><br /> |
- | BL21-JS19HisFokA | + | <be>BL21-JS119StrepFokA</be><br /> |
- | | + | <be>BL21-JS118Strep</be><br /> |
- | - Plated on KM plats<br> | + | <be>BL21-JS118HisFokA</be><br /> |
- | | + | <be>BL21-JS19HisFokA |
- | <br>* Preparation of overnight cultures 5 ml LB medium each | + | - Plated on KM plats</be></small><br /> |
- | - pBad+Kan<br> | + | </div> |
- | -pET39B(+)<br>*Amp<br> | + | <div style="margin-left: 80px;"> |
- | --> Stored in 37°C room | + | <be></be></div> |
- | | + | <div style="margin-left: 40px;"><be></be><br /> |
- | <br>*Digest: | + | <be></be></div> |
- | - pMASplitFoka<br> | + | <ul style="margin-left: 40px;"> |
- | - pMAFoka<br> | + | <li><be> Preparation of overnight cultures 5 ml |
- | - pMAFoki<br> | + | LB medium each |
- | - pMASplitFoki<br> | + | - pBad+Kan</be></li> |
- | - pMALongLiFoka<br> | + | </ul> |
- | - pMALongliFoki<br> | + | <div style="margin-left: 80px;"><small><be>-pET39B(+)</be></small><br /> |
- | - pMAShortli<br> | + | <be></be></div> |
- | - pMAMiddleli<br> | + | <ul style="margin-left: 40px;"> |
- | - pMALongli<br> | + | <li><be>Amp</be></li> |
- | - pExStrepFluA<br> | + | </ul> |
- | | + | <div style="margin-left: 80px;"><small><be>--> |
- | | + | Stored in 37°C room |
- | <br>*preparative gel | + | </be></small><br /> |
- | [[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA<|400x400px]] | + | <be></be></div> |
- | ->interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly | + | <ul style="margin-left: 40px;"> |
- | <br>*gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09 | + | <li><be>Digest: |
- | | + | - pMASplitFoka</be></li> |
- | | + | </ul> |
- | <br>* digest and ligation into new pMA:<br> | + | <div style="margin-left: 40px;"><small><be>- |
- | -pMASplitFokA clone1 19.08.09<br> | + | pMAFoka</be><br /> |
- | -pMAFokA clone2 05.08.09<br> | + | <be>- pMAFoki</be><br /> |
- | -pMAFoki clone2 05.08.09<br> | + | <be>- pMASplitFoki</be><br /> |
- | -pMASplitFoki clone2 06.08.09<br> | + | <be>- pMALongLiFoka</be><br /> |
- | -pMAshortLi clone2 10.09.09<br> | + | <be>- pMALongliFoki</be><br /> |
- | -pMAmiddelLi clone2 10.09.09<br> | + | <be>- pMAShortli</be><br /> |
- | -pMAlongLi clone1 10.09.09<br> | + | <be>- pMAMiddleli</be><br /> |
- | -pMAlongLiFokA clone1 10.09.09<br> | + | <be>- pMALongli</be></small><br /> |
- | -pMAlongLiFoki clone1 10.09.09<br> | + | <small><be>- pExStrepFluA</be><br /> |
- | digestion with EcoRI and SpeI (vector and insert) | + | </small></div> |
- | <br> | + | <div style="margin-left: 80px;"> |
- | <br>* Transformation of:<br> | + | <small><be></be></small></div> |
- | -pMAFokA clone2 05.08.09<br> | + | <div style="margin-left: 40px;"><be></be><br /> |
- | -pMAFoki clone2 05.08.09<br> | + | <be></be></div> |
- | -pMASplitFoki clone2 06.08.09<br> | + | <ul style="margin-left: 40px;"> |
- | | + | <li><be>preparative gel |
- | <br>*plasmidprep of:<br> | + | [[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel; |
- | 1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick<br> | + | Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, |
- | 2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick<br> | + | 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, |
- | 3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick<br><br> | + | 9.pMALongli, 10.pExStrepFluA<|400x400px]] |
- | | + | ->interpretation: just the linker resulted in too short |
- | 4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick <br> | + | fragments, |
- | 5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick<br> | + | thus the hybridized linkers have to be cloned in pMA directly |
- | 6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick<br><br> | + | </be></li> |
- | | + | <li><be>gelextraction of digest from 02.10.09 and also |
- | 7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit1<br> | + | pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from |
- | 8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit2<br> | + | 01.10.09 |
- | 9. pEX_DsbA+Strep+Dig+Split+FokA Clone3<br><br> | + | </be></li> |
- | | + | <li><be>digest and ligation into new pMA:</be></li> |
- | 10. pEX_DsbA+His+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit3<br> | + | </ul> |
- | 11. pEX_DsbA+His+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit4<br> | + | <div style="margin-left: 40px;"><small><be>-pMASplitFokA |
- | 12. pEX_DsbA+His+Dig+Split+FokA Clone3<br><br> | + | clone1 19.08.09</be><br /> |
- | | + | <be>-pMAFokA clone2 05.08.09</be><br /> |
- | <br>*testdigest of: | + | <be>-pMAFoki clone2 05.08.09</be><br /> |
- | V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick<br> | + | <be>-pMASplitFoki clone2 06.08.09</be><br /> |
- | V2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick<br> | + | <be>-pMAshortLi clone2 10.09.09</be><br /> |
- | V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick<br><br> | + | <be>-pMAmiddelLi clone2 10.09.09</be><br /> |
- | | + | <be>-pMAlongLi clone1 10.09.09</be><br /> |
- | V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick <br> | + | <be>-pMAlongLiFokA clone1 10.09.09</be><br /> |
- | V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick<br> | + | <be>-pMAlongLiFoki clone1 10.09.09</be><br /> |
- | V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick<br><br> | + | <be>digestion with EcoRI and SpeI (vector and insert) |
- | | + | </be></small><br /> |
- | V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 <br> | + | </div> |
- | V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 <br> | + | <div style="margin-left: 80px;"> |
- | V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3<br><br> | + | <be></be></div> |
- | | + | <div style="margin-left: 40px;"><be></be><br /> |
- | V10. pEX_DsbA+His+Dig+Split+FokA Clone1 <br> | + | <be></be></div> |
- | V11. pEX_DsbA+His+Dig+Split+FokA Clone2 <br> | + | <ul style="margin-left: 40px;"> |
- | V12. pEX_DsbA+His+Dig+Split+FokA Clone3<br><br> | + | <li><be> Transformation of:</be></li> |
- | | + | </ul> |
- | V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)<br> | + | <div style="margin-left: 40px;"><small><be>-pMAFokA |
- | V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)<br> | + | clone2 |
- | with NcoI-HF and XbaI 1h at 37°C<br> | + | 05.08.09</be><br /> |
- | | + | <be> -pMAFoki clone2 05.08.09</be><br /> |
- | --> geldbild
| + | <be></be><be>-pMASplitFoki |
- | | + | clone2 06.08.09</be></small><br /> |
- | <h3>03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph </h3> | + | <be></be><be></be><br /> |
- | <br>*plasmid prep of pET and pBad and Glytsocks(Xbl) | + | <be></be></div> |
- | <br>*new aliquots Ampicilin (70%EtOH) | + | <ul style="margin-left: 40px;"> |
- | <br>*Test digest M13dsDNA+fokI as control for dsDNA , the pictures showed the two expected lanes(hardly visible on the printout). | + | <li><be>plasmidprep of:</be></li> |
- | | + | </ul> |
- | [[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel; M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09, M13dsDNA02.10.09 digest fokI |400x400px]] | + | <div style="margin-left: 40px;"><small><be>1. |
- | <br>*digest of | + | pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be><br /> |
- | - pEx-Strep-Dig-LongLinker-Fok(inactive)<br> | + | <be>2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br /> |
- | - pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br> | + | <be>3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be><br /> |
- | - pEx-Strep-Dig-ShortLinker-Fok(inactive)<br> | + | <be></be><br /> |
- | - pEx-Strep-Dig-SplitLinker-Fok(inactive)<br> | + | <be>4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick </be><br /> |
- | - pEx-His-Dig-SplitLinker_Fok(inactive)<br> | + | <be>5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick</be><br /> |
- | - pEx-His-Dig-SplitLinker_Fok(active)<br> | + | <be>6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick</be><br /> |
- | - pEx-Strep-Dig-SplitLinker-Fok(active)<br> | + | <be></be><br /> |
- | - pEx-His-Dig-LongLinker-Fok(inactive)<br> | + | <be>7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --> seq |
- | - pEx-His-FluA-LongLinker-Fok(inactive)<br> | + | 02.10.09 Gerrit1</be><br /> |
- | - pEx-His-FluA-MiddleLinker-Fok(inactive)<br> | + | <be>8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --> seq |
- | - pEx-His-FluA-ShortLinker-Fok(inactive)<br> | + | 02.10.09 Gerrit2</be><br /> |
- | - pEx-His-FluA-SplitLinker-Fok(inactive)<br> | + | <be>9. pEX_DsbA+Strep+Dig+Split+FokA Clone3</be><br /> |
- | - pEx-His-Dig<br> | + | <be></be><br /> |
- | - pMA<br> | + | <be>10. pEX_DsbA+His+Dig+Split+FokA Clone1 --> seq |
| + | 02.10.09 Gerrit3</be><br /> |
| + | <be>11. pEX_DsbA+His+Dig+Split+FokA Clone2 --> seq |
| + | 02.10.09 Gerrit4</be><br /> |
| + | <be>12. pEX_DsbA+His+Dig+Split+FokA Clone3</be></small><br /> |
| + | <be></be><be></be><br /> |
| + | <be></be><br /> |
| + | <be></be></div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li><be>testdigest of: |
| + | V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be></li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small><be>V2. |
| + | pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br /> |
| + | <be>V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be><br /> |
| + | <be></be><br /> |
| + | <be>V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick </be><br /> |
| + | <be>V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick</be><br /> |
| + | <be>V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick</be><br /> |
| + | <be></be><br /> |
| + | <be>V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 </be><br /> |
| + | <be>V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 </be><br /> |
| + | <be>V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3</be><br /> |
| + | <be></be><br /> |
| + | <be>V10. pEX_DsbA+His+Dig+Split+FokA Clone1 </be><br /> |
| + | <be>V11. pEX_DsbA+His+Dig+Split+FokA Clone2 </be><br /> |
| + | <be>V12. pEX_DsbA+His+Dig+Split+FokA Clone3</be><br /> |
| + | <be></be><br /> |
| + | <be>V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)</be><br /> |
| + | <be>V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)</be><br /> |
| + | <be>with NcoI-HF and XbaI 1h at 37°C</be><br /> |
| + | </small><be><small> --> geldbild</small> |
| + | </be></div> |
| + | <h3 style="margin-left: 40px;">03.10.09 Manu, Hannes, |
| + | Gerrit, Caro, Timo, Christoph </h3> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>plasmid prep of pET and pBad and Glytsocks(Xbl) |
| + | </li> |
| + | <li>new aliquots Ampicilin (70%EtOH) |
| + | </li> |
| + | <li>Test digest M13dsDNA+fokI as control for dsDNA , the |
| + | pictures |
| + | showed the two expected lanes(hardly visible on the printout). |
| + | [[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel; |
| + | M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09, |
| + | M13dsDNA02.10.09 digest fokI |400x400px]] |
| + | </li> |
| + | </ul> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br /> |
| + | - pEx-Strep-Dig-ShortLinker-Fok(inactive)<br /> |
| + | - pEx-Strep-Dig-SplitLinker-Fok(inactive)<br /> |
| + | - pEx-His-Dig-SplitLinker_Fok(inactive)<br /> |
| + | - pEx-His-Dig-SplitLinker_Fok(active)<br /> |
| + | - pEx-Strep-Dig-SplitLinker-Fok(active)<br /> |
| + | - pEx-His-Dig-LongLinker-Fok(inactive)<br /> |
| + | - pEx-His-FluA-LongLinker-Fok(inactive)<br /> |
| + | - pEx-His-FluA-MiddleLinker-Fok(inactive)<br /> |
| + | - pEx-His-FluA-ShortLinker-Fok(inactive)<br /> |
| + | - pEx-His-FluA-SplitLinker-Fok(inactive)<br /> |
| + | - pEx-His-Dig<br /> |
| + | - pMA<br /> |
| with NgoMIV and SpeI | | with NgoMIV and SpeI |
- | <br>*digest of | + | </small><br /> |
- | - pMA<br> | + | </div> |
- | with XbaI and AgeI | + | <ul style="margin-left: 40px;"> |
- | | + | <li>digest of |
- | <br>*Ligation of: | + | - pMA with XbaI and AgeI |
- | - Strep-Dig-LongLinker-Fok(inactive)<br> | + | </li> |
- | - Strep-Dig-MiddleLinker-Fok(inactive)<br> | + | </ul> |
- | - trep-Dig-ShortLinker-Fok(inactive)<br> | + | <ul style="margin-left: 40px;"> |
- | - Strep-Dig-SplitLinker-Fok(inactive)<br> | + | <li>Ligation of: - Strep-Dig-LongLinker-Fok(inactive)</li> |
- | - His-Dig-SplitLinker_Fok(inactive)<br> | + | </ul> |
- | - His-Dig-SplitLinker_Fok(active)<br> | + | <div style="margin-left: 40px;"><small>- |
- | - Strep-Dig-SplitLinker-Fok(active)<br> | + | Strep-Dig-MiddleLinker-Fok(inactive)<br /> |
- | - His-Dig-LongLinker-Fok(inactive)<br> | + | - trep-Dig-ShortLinker-Fok(inactive)<br /> |
- | - His-FluA-LongLinker-Fok(inactive)<br> | + | - Strep-Dig-SplitLinker-Fok(inactive)<br /> |
- | - His-FluA-MiddleLinker-Fok(inactive)<br> | + | - His-Dig-SplitLinker_Fok(inactive)<br /> |
- | - His-FluA-ShortLinker-Fok(inactive)<br> | + | - His-Dig-SplitLinker_Fok(active)<br /> |
- | - His-FluA-SplitLinker-Fok(inactive)<br> | + | - Strep-Dig-SplitLinker-Fok(active)<br /> |
- | into pMA<br> | + | - His-Dig-LongLinker-Fok(inactive)<br /> |
- | | + | - His-FluA-LongLinker-Fok(inactive)<br /> |
- | and<br> | + | - His-FluA-MiddleLinker-Fok(inactive)<br /> |
- | - Strep-Dig-LongLinker-Fok(inactive)<br> | + | - His-FluA-ShortLinker-Fok(inactive)<br /> |
- | - Strep-Dig-MiddleLinker-Fok(inactive)<br> | + | - His-FluA-SplitLinker-Fok(inactive)<br /> |
- | - trep-Dig-ShortLinker-Fok(inactive)<br> | + | into pMA<br /> |
| + | and<br /> |
| + | - Strep-Dig-LongLinker-Fok(inactive)<br /> |
| + | - Strep-Dig-MiddleLinker-Fok(inactive)<br /> |
| + | - trep-Dig-ShortLinker-Fok(inactive)<br /> |
| into new pEX | | into new pEX |
- | | + | </small><br /> |
- | <br>*inoculation of: | + | </div> |
- | -pMASplitFokA clone1 19.08.09<br> | + | <ul style="margin-left: 40px;"> |
- | -pMAFokA clone2 05.08.09 (new)<br> | + | <li>inoculation of: |
- | -pMAFoki clone2 05.08.09 (new)<br> | + | -pMASplitFokA clone1 19.08.09</li> |
- | -pMASplitFoki clone2 06.08.09 (new)<br> | + | </ul> |
- | -pMAshortLi clone2 10.09.09<br> | + | <div style="margin-left: 40px;"><small>-pMAFokA |
- | -pMAmiddelLi clone2 10.09.09<br> | + | clone2 05.08.09 |
- | -pMAlongLi clone1 10.09.09<br> | + | (new)<br /> |
- | -pMAlongLiFokA clone1 10.09.09<br> | + | -pMAFoki clone2 05.08.09 (new)<br /> |
- | -pMAlongLiFoki clone1 10.09.09<br><br> | + | -pMASplitFoki clone2 06.08.09 (new)<br /> |
- | -pMAFokA clone2 05.08.09 (old)<br> | + | -pMAshortLi clone2 10.09.09<br /> |
- | -pMAFoki clone2 05.08.09 (old)<br> | + | -pMAmiddelLi clone2 10.09.09<br /> |
- | -pMASplitFoki clone2 06.08.09 (old)<br> | + | -pMAlongLi clone1 10.09.09<br /> |
- | | + | -pMAlongLiFokA clone1 10.09.09<br /> |
- | <br>*inoculation and plasmid preperation: | + | -pMAlongLiFoki clone1 10.09.09<br /> |
| + | <br /> |
| + | -pMAFokA clone2 05.08.09 (old)<br /> |
| + | -pMAFoki clone2 05.08.09 (old)<br /> |
| + | -pMASplitFoki clone2 06.08.09 (old)</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>inoculation and plasmid preperation: |
| -pEX_strepFluA | | -pEX_strepFluA |
- | <br>*Quenching test with HisFluASplitFoki and fluorescin-tagged oligos | + | </li> |
- | <br>*BB Ago Pcr via Taq | + | <li>Quenching test with HisFluASplitFoki and fluorescin-tagged |
- | <br>*Started makin new VCS M13 Phages see Protocol day 1 | + | oligos |
- | | + | </li> |
- | <h3>Plan for 04.10.09</h3> | + | <li>BB Ago Pcr via Taq |
- | <br>*inoculation of | + | </li> |
- | - pMA-Strep-Dig-LongLinker-Fok(inactive)<br> | + | <li>Started makin new VCS M13 Phages see Protocol day 1 |
- | - pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br> | + | </li> |
- | - pMA-Strep-Dig-ShortLinker-Fok(inactive)<br> | + | </ul> |
- | - pMA-Strep-Dig-SplitLinker-Fok(inactive)<br> | + | <h3 style="margin-left: 40px;">Plan for 04.10.09</h3> |
- | - pMA-His-Dig-SplitLinker_Fok(inactive)<br> | + | <div style="margin-left: 40px;"><br /> |
- | - pMA-His-Dig-SplitLinker_Fok(inactive)<br> | + | </div> |
- | - pMA-Strep-Dig-SplitLinker-Fok(active)<br> | + | <ul style="margin-left: 40px;"> |
- | - pMA-His-Dig-LongLinker-Fok(inactive)<br> | + | <li>inoculation of |
- | - pMA-His-FluA-LongLinker-Fok(inactive)<br> | + | - pMA-Strep-Dig-LongLinker-Fok(inactive)</li> |
- | - pMA-His-FluA-MiddleLinker-Fok(inactive)<br> | + | </ul> |
- | - pMA-His-FluA-ShortLinker-Fok(inactive)<br> | + | <div style="margin-left: 40px;"><small>- |
- | - pMA-His-FluA-SplitLinker-Fok(inactive)<br> | + | pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br /> |
| + | - pMA-Strep-Dig-ShortLinker-Fok(inactive)<br /> |
| + | - pMA-Strep-Dig-SplitLinker-Fok(inactive)<br /> |
| + | - pMA-His-Dig-SplitLinker_Fok(inactive)<br /> |
| + | - pMA-His-Dig-SplitLinker_Fok(inactive)<br /> |
| + | - pMA-Strep-Dig-SplitLinker-Fok(active)<br /> |
| + | - pMA-His-Dig-LongLinker-Fok(inactive)<br /> |
| + | - pMA-His-FluA-LongLinker-Fok(inactive)<br /> |
| + | - pMA-His-FluA-MiddleLinker-Fok(inactive)<br /> |
| + | - pMA-His-FluA-ShortLinker-Fok(inactive)<br /> |
| + | - pMA-His-FluA-SplitLinker-Fok(inactive)<br /> |
| into LB-Amp | | into LB-Amp |
- | <br>*digestion of pEx-Strep-Flua with AgeI and PstI | + | </small><br /> |
- | <br>*digestion of pMA-Short/Middle/Long/Split-Fok(active and inactive) | + | </div> |
- | <br>*ligation of the above parts and transformation into RV308 | + | <ul style="margin-left: 40px;"> |
- | <br>*plasmid preparation of... | + | <li>digestion of pEx-Strep-Flua with AgeI and PstI |
- | | + | </li> |
- | -pMASplitFokA <br> | + | <li>digestion of pMA-Short/Middle/Long/Split-Fok(active and |
- | -pMAFokA <br> | + | inactive) |
- | -pMAFoki <br> | + | </li> |
- | -pMASplitFoki <br> | + | <li>ligation of the above parts and transformation into RV308 |
- | -pMAshortLi<br> | + | </li> |
- | -pMAmiddelLi<br> | + | <li>plasmid preparation of... |
- | -pMAlongLi <br> | + | -pMASplitFokA </li> |
- | -pMAlongLiFokA <br> | + | </ul> |
- | -pMAlongLiFoki <br> | + | <div style="margin-left: 40px;"><small>-pMAFokA <br /> |
- | -pMAFokA (old)<br> | + | -pMAFoki <br /> |
- | -pMAFoki (old)<br> | + | -pMASplitFoki <br /> |
- | -pMASplitFoki (old)<br> | + | -pMAshortLi<br /> |
- | | + | -pMAmiddelLi<br /> |
- | <h3>04.10.09, Laura, Anika, Max</h3> | + | -pMAlongLi <br /> |
- | <br>* Plasmid preparation of | + | -pMAlongLiFokA <br /> |
- | -pMASplitFokA <br> | + | -pMAlongLiFoki <br /> |
- | -pMAFokA <br> | + | -pMAFokA (old)<br /> |
- | -pMAFoki <br> | + | -pMAFoki (old)<br /> |
- | -pMASplitFoki <br> | + | -pMASplitFoki (old)</small><br /> |
- | -pMAshortLiFokA<br> | + | </div> |
- | -pMAshortLiFoki<br> | + | <h3 style="margin-left: 40px;">04.10.09, Laura, Anika, Max</h3> |
- | -pMAmiddelLiFokA<br> | + | <div style="margin-left: 40px;"><br /> |
- | -pMAmiddelLiFoki<br> | + | </div> |
- | -pMAlongLiFokA <br> | + | <ul style="margin-left: 40px;"> |
- | -pMAlongLiFoki <br> | + | <li> Plasmid preparation of -pMASplitFokA </li> |
- | -pMAFokA (old)<br> | + | </ul> |
- | -pMAFoki (old)<br> | + | <div style="margin-left: 40px;"><small>-pMAFokA <br /> |
- | -pMASplitFoki (old)<br> | + | -pMAFoki <br /> |
- | clone 1 and 2,respectively - results see notebook<br> | + | -pMASplitFoki <br /> |
- | <br>* Glycerolstock of <br> | + | -pMAshortLiFokA<br /> |
- | -pMASplitFokA <br> | + | -pMAshortLiFoki<br /> |
- | -pMAFokA <br> | + | -pMAmiddelLiFokA<br /> |
- | -pMAFoki <br> | + | -pMAmiddelLiFoki<br /> |
- | -pMASplitFoki <br> | + | -pMAlongLiFokA <br /> |
- | -pMAshortLiFokA<br> | + | -pMAlongLiFoki <br /> |
- | -pMAshortLiFoki<br> | + | -pMAFokA (old)<br /> |
- | -pMAmiddelLiFokA<br> | + | -pMAFoki (old)<br /> |
- | -pMAmiddelLiFoki<br> | + | -pMASplitFoki (old)<br /> |
- | -pMAlongLiFokA <br> | + | clone 1 and 2,respectively - results see notebook |
- | -pMAlongLiFoki <br> | + | </small><br /> |
- | -pMAFokA (old)<br> | + | </div> |
- | -pMAFoki (old)<br> | + | <ul style="margin-left: 40px;"> |
- | -pMASplitFoki (old)<br> | + | <li> Glycerolstock of </li> |
- | clone 3 | + | </ul> |
- | <br>* Pellets of<br> | + | <div style="margin-left: 40px;"><small>-pMASplitFokA |
- | -pMASplitFokA <br> | + | <br /> |
- | -pMAFokA <br> | + | -pMAFokA <br /> |
- | -pMAFoki <br> | + | -pMAFoki <br /> |
- | -pMASplitFoki <br> | + | -pMASplitFoki <br /> |
- | -pMAshortLiFokA<br> | + | -pMAshortLiFokA<br /> |
- | -pMAshortLiFoki<br> | + | -pMAshortLiFoki<br /> |
- | -pMAmiddelLiFokA<br> | + | -pMAmiddelLiFokA<br /> |
- | -pMAmiddelLiFoki<br> | + | -pMAmiddelLiFoki<br /> |
- | -pMAlongLiFokA <br> | + | -pMAlongLiFokA <br /> |
- | -pMAlongLiFoki <br> | + | -pMAlongLiFoki <br /> |
- | -pMAFokA (old)<br> | + | -pMAFokA (old)<br /> |
- | -pMAFoki (old)<br> | + | -pMAFoki (old)<br /> |
- | -pMASplitFoki (old)<br> | + | -pMASplitFoki (old)</small><br /> |
- | clone 1 and 2,respectively - stored in Pelletbox, -20°C | + | <small>clone 3 |
- | <br>*Digest of | + | </small><br /> |
- | 1. pEXHisDigMiddleLFoki | + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Pellets of</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-pMASplitFokA |
| + | <br /> |
| + | -pMAFokA <br /> |
| + | -pMAFoki <br /> |
| + | -pMASplitFoki <br /> |
| + | -pMAshortLiFokA<br /> |
| + | -pMAshortLiFoki<br /> |
| + | -pMAmiddelLiFokA<br /> |
| + | -pMAmiddelLiFoki<br /> |
| + | -pMAlongLiFokA <br /> |
| + | -pMAlongLiFoki <br /> |
| + | -pMAFokA (old)<br /> |
| + | -pMAFoki (old)<br /> |
| + | -pMASplitFoki (old)<br /> |
| + | clone 1 and 2,respectively |
| + | - stored in Pelletbox, -20°C |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Digest of 1. pEXHisDigMiddleLFoki |
| 2. pMaScFaantiNIP | | 2. pMaScFaantiNIP |
| 3. prep of Ligation pEX+CAT of 09.07.09 old | | 3. prep of Ligation pEX+CAT of 09.07.09 old |
- | 4. pMAShortLFoka<br> | + | 4. pMAShortLFoka</li> |
- | 5. pMAShortLFoki<br> | + | </ul> |
- | 6. pMAMiddleLFoka<br> | + | <div style="margin-left: 40px;"><small>5. |
- | 7. pMAMiddleLFoki<br> | + | pMAShortLFoki<br /> |
- | 8. pMALongLFoka<br> | + | 6. pMAMiddleLFoka<br /> |
- | 9. pMALongLFoki<br> | + | 7. pMAMiddleLFoki<br /> |
- | 10.pEXStrepFlua<br> | + | 8. pMALongLFoka<br /> |
- | <br>*Preparative Gel of Digest<br> | + | 9. pMALongLFoki<br /> |
- | [[Image:Freiburg09_041009_umklonierung_gel.JPG|none|thumb|Agarose gel; Lanes: 1. pEXHisDigMiddleLFoki, 2. pMaScFaantiNIP, 3. pEX+CAT, 4. pMAShortLFoka, 5. pMAShortLFoki, 6. pMAMiddleLFoka, 7. pMAMiddleLFoki, 8. pMALongLFoka, 9. pMALongLFoki,10.pEXStrepFlua|400x400px]]
| + | 10.pEXStrepFlua</small><br /> |
- | Freiburg09_041009_umklonierung_gel.JPG
| + | <br /> |
- | <br>*Gelextraction<br> | + | </div> |
- | <br>*Ligation of<br> | + | <ul style="margin-left: 40px;"> |
- | 1.pMAHisDigMiddleFoki<br> | + | <li>Preparative Gel of Digest</li> |
- | 2.pMACAT<br> | + | </ul> |
- | 3.pEXStrepFluAShortLFokA<br> | + | <table style="text-align: center; width: 750px; margin-left: 40px;" |
- | 4.pEXStrepFluAShortLFoki<br> | + | border="0" cellpadding="0" cellspacing="0"> |
- | 5.pEXStrepFluAmiddleLFokA<br> | + | <tbody> |
- | 6.pEXStrepFluAMiddleLFoki<br> | + | <tr> |
- | 7.pEXStrepFluALongLFokA<br> | + | <td align="center"><img style="width: 380px; height: 288px;" |
- | 8.pEXStrepFluALongLFoki<br> | + | alt="" |
- | 9.JS 419StrepDigSplitFokA<br> | + | src="https://static.igem.org/mediawiki/2009/d/df/Freiburg09_021009_umklonierung.JPG" /></td> |
- | 10.JS 419HisDigSplitFokA<br> | + | </tr> |
- | 11.JS 418HisDigSplitFokA<br> | + | <tr> |
- | Approach: 8µl H2O, 3 µl Vector, 6µl Insert, 2µl Quick Ligase Buffer, 1 µl Quick Ligase, 15 min, Room Temp.<br> | + | <td><small></small><small>Agarose gel; |
- | <br>*Dephosphorylation of pBAD Vector: <br> | + | Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, |
- | Approach: 2µl Eluat, 3,1 µl Fast Ap Buffer 10x, 1µl Fast Ap, 10 min. 37°C, 5 min. 75°C<br> | + | 4.pMASplitFoki, |
- | <br>*Transformation of<br> | + | 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, |
- | 1.pMAHisDigMiddleFoki (RV)<br> | + | 9.pMALongli, 10.pExStrepFluA</small></td> |
- | 2.pMACAT(RV)<br> | + | </tr> |
- | 3.pEXStrepFluAShortLFokA(RV)<br> | + | </tbody> |
- | 4.pEXStrepFluAShortLFoki(RV)<br> | + | </table> |
- | 5.pEXStrepFluAmiddleLFokA(RV)<br> | + | <div style="margin-left: 40px;"><br /> |
- | 6.pEXStrepFluAMiddleLFoki(RV)<br> | + | <br /> |
- | 7.pEXStrepFluALongLFokA(RV)<br> | + | </div> |
- | 8.pEXStrepFluALongLFoki(RV)<br> | + | <ul style="margin-left: 40px;"> |
- | 9.pBAD (RV)<br> | + | <li>Gelextraction</li> |
- | 10.pJS 419StrepDigSplitFokA(XBL)<br> | + | <li>Ligation of</li> |
- | 11.pJS 419HisDigSplitFokA(XBL)<br> | + | </ul> |
- | 12.pJS 418HisDigSplitFokA(XBL)<br> | + | <div style="margin-left: 40px;"><small> |
- | <br>*test digest of plasmid preps from today | + | 1.pMAHisDigMiddleFoki<br /> |
- | -> run on a gel with digest pET39, xbaI, ecoRI from Tobi | + | 2.pMACAT<br /> |
- | <br>*starter culture of pEx_DsbA_HisDigSplitFoka | + | 3.pEXStrepFluAShortLFokA<br /> |
- | -> modified expression has to be done tomorrow | + | 4.pEXStrepFluAShortLFoki<br /> |
- | <br>*inoculation of pJS418 and pJS419 for glycerolstocks tomorrow | + | 5.pEXStrepFluAmiddleLFokA<br /> |
- | | + | 6.pEXStrepFluAMiddleLFoki<br /> |
- | <br>*Taq AGO BB PCR did not work | + | 7.pEXStrepFluALongLFokA<br /> |
- | | + | 8.pEXStrepFluALongLFoki<br /> |
- | <br>*Phageproduction day 2 | + | 9.JS 419StrepDigSplitFokA<br /> |
- | | + | 10.JS 419HisDigSplitFokA<br /> |
- | <h3>05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika</h3> | + | 11.JS 418HisDigSplitFokA<br /> |
- | | + | Approach: 8µl H2O, 3 µl Vector, 6µl |
- | <br>* 3l LB and 2L LB Agar | + | Insert, 2µl Quick Ligase Buffer, 1 µl Quick Ligase, |
- | | + | 15 min, Room Temp.<br /> |
- | <br>*Periplasma Project | + | </small><br /> |
- | | + | </div> |
- | <br>*digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09) | + | <ul style="margin-left: 40px;"> |
- | <br>*test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka | + | <li>Dephosphorylation of pBAD Vector: </li> |
- | <br>*1) Gelextraction<br> | + | </ul> |
- | | + | <div style="margin-left: 40px;"><small>Approach: |
- | --> Gelbild <br> | + | 2µl |
- | | + | Eluat, 3,1 µl Fast Ap Buffer 10x, 1µl Fast Ap, 10 |
- | 1) Vector: pEXHisFluSplitFoki<br> | + | min. 37°C, 5 min. 75°C</small><br /> |
- | 2)Insert: RBSStrepDigSplitFokA<br> | + | <br /> |
- | 3)PET39b+<br> | + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | <br>*2)Ligation<br> | + | <li>Transformation of</li> |
- | -10ul 2 fold buffer<br> | + | </ul> |
- | - 6 µl Insert<br> | + | <div style="margin-left: 40px;"><small>1.pMAHisDigMiddleFoki |
- | -3µl Vector<br> | + | (RV)<br /> |
- | -1µl Quick Ligase<br> | + | 2.pMACAT(RV)<br /> |
- | | + | 3.pEXStrepFluAShortLFokA(RV)<br /> |
- | --> 15 min at RT<br> | + | 4.pEXStrepFluAShortLFoki(RV)<br /> |
- | | + | 5.pEXStrepFluAmiddleLFokA(RV)<br /> |
- | Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA<br> | + | 6.pEXStrepFluAMiddleLFoki(RV)<br /> |
- | | + | 7.pEXStrepFluALongLFokA(RV)<br /> |
- | <br>*3)Transformation<br> | + | 8.pEXStrepFluALongLFoki(RV)<br /> |
- | -5 µl DNA of Ligation + BL21d3 and XBL | + | 9.pBAD (RV)<br /> |
- | | + | 10.pJS 419StrepDigSplitFokA(XBL)<br /> |
- | <br>*'''Mini prep with with Qiagen Spin Miniprep Kit of''' | + | 11.pJS 419HisDigSplitFokA(XBL)<br /> |
- | | + | 12.pJS 418HisDigSplitFokA(XBL)</small><br /> |
- | - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3<br> | + | <br /> |
- | - pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br> | + | </div> |
- | - pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br> | + | <ul style="margin-left: 40px;"> |
- | - pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br> | + | <li>test digest of plasmid preps from today |
- | - pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br> | + | -> run on a gel with digest pET39, xbaI, ecoRI from Tobi |
- | - pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br> | + | </li> |
- | - pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br> | + | <li>starter culture of pEx_DsbA_HisDigSplitFoka |
- | - pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br> | + | -> modified expression has to be done tomorrow |
- | - pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br> | + | </li> |
- | - pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br> | + | <li>inoculation of pJS418 and pJS419 for glycerolstocks |
- | - pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br> | + | tomorrow |
- | - pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br> | + | </li> |
| + | <li>Taq AGO BB PCR did not work |
| + | </li> |
| + | <li>Phageproduction day 2 |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;">05.10.09, Laura, Manu, |
| + | Julia, Christophe, Caro, Hannes, |
| + | Gerrit, Anika</h3> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> 3l LB and 2L LB Agar |
| + | </li> |
| + | <li>Periplasma Project |
| + | </li> |
| + | <li>digest pExHisFluASplitFoki (prep from 24.08.09) and |
| + | RBS_StrepDigSplitFoka (prep from 29.09.09) |
| + | </li> |
| + | <li>test digests of pMAFoka, pMALongliFoki, pMAFoka old, |
| + | pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka |
| + | </li> |
| + | <li>1) Gelextraction</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>--> |
| + | Gelbild <br /> |
| + | 1) Vector: pEXHisFluSplitFoki<br /> |
| + | 2)Insert: RBSStrepDigSplitFokA<br /> |
| + | 3)PET39b+</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>2)Ligation</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-10ul 2 fold |
| + | buffer<br /> |
| + | - 6 µl Insert<br /> |
| + | -3µl Vector<br /> |
| + | -1µl Quick Ligase<br /> |
| + | --> 15 min at RT<br /> |
| + | Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>3)Transformation</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-5 |
| + | µl DNA of |
| + | Ligation + BL21d3 and XBL |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>'''Mini prep with with Qiagen Spin Miniprep Kit of''' |
| + | - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br /> |
| + | - pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br /> |
| + | - pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br /> |
| + | - pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br /> |
| - pMA-Dbsa 1,2,3 | | - pMA-Dbsa 1,2,3 |
- | | + | </small><br /> |
- | <br>*'''Made Glycerolstocks of''' | + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | <br>*700µl Cellsuspension+ 300µl Glycerol | + | <li>Made Glycerolstocks of |
- | - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3<br> | + | </li> |
- | - pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br> | + | <li>700µl Cellsuspension+ 300µl Glycerol |
- | - pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br> | + | - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li> |
- | - pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br> | + | </ul> |
- | - pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br> | + | <div style="margin-left: 40px;"><small>- |
- | - pMA-His-Dig-SplitLinker_Fok(active)1,2,3<br> | + | pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br /> |
- | - pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br> | + | - pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br /> |
- | - pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br> | + | - pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br /> |
- | - pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br> | + | - pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br /> |
- | - pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br> | + | - pMA-His-Dig-SplitLinker_Fok(active)1,2,3<br /> |
- | - pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br> | + | - pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br /> |
- | - pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br> | + | - pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br /> |
| + | - pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br /> |
| - pMA-Dbsa 1,2,3 | | - pMA-Dbsa 1,2,3 |
- | | + | </small><br /> |
- | <br>*'''Test Digestion of''' | + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | <br>*put in each sample: 5µl DNA, 2,5µl H2O, 1µl Buffer 3, 1µl EcoRI, 1µl PstI, 1µl BSA | + | <li>'''Test Digestion of''' |
- | | + | </li> |
- | - pMA-Strep-Dig-LongLinker-Fok(inactive)3<br> | + | <li>put in each sample: 5µl DNA, 2,5µl H2O, |
- | - pMA-Strep-Dig-MiddleLinker-Fok(inactive)1<br> | + | 1µl Buffer 3, 1µl EcoRI, 1µl PstI, |
- | - pMA-Strep-Dig-ShortLinker-Fok(inactive)1<br> | + | 1µl BSA |
- | - pMA-Strep-Dig-SplitLinker-Fok(inactive)3<br> | + | - pMA-Strep-Dig-LongLinker-Fok(inactive)3</li> |
- | - pMA-His-Dig-SplitLinker_Fok(inactive)1<br> | + | </ul> |
- | - pMA-His-Dig-SplitLinker_Fok(active)2<br> | + | <div style="margin-left: 40px;"><small>- |
- | - pMA-Strep-Dig-SplitLinker-Fok(active)1<br> | + | pMA-Strep-Dig-MiddleLinker-Fok(inactive)1<br /> |
- | - pMA-His-Dig-LongLinker-Fok(inactive)1<br> | + | - pMA-Strep-Dig-ShortLinker-Fok(inactive)1<br /> |
- | - pMA-His-FluA-LongLinker-Fok(inactive)3<br> | + | - pMA-Strep-Dig-SplitLinker-Fok(inactive)3<br /> |
- | - pMA-His-FluA-MiddleLinker-Fok(inactive)3<br> | + | - pMA-His-Dig-SplitLinker_Fok(inactive)1<br /> |
- | - pMA-His-FluA-ShortLinker-Fok(inactive)1<br> | + | - pMA-His-Dig-SplitLinker_Fok(active)2<br /> |
- | - pMA-His-FluA-SplitLinker-Fok(inactive)2<br> | + | - pMA-Strep-Dig-SplitLinker-Fok(active)1<br /> |
- | - pMA-Dbsa 2<br> | + | - pMA-His-Dig-LongLinker-Fok(inactive)1<br /> |
- | | + | - pMA-His-FluA-LongLinker-Fok(inactive)3<br /> |
- | <br>* Transformation of<br> | + | - pMA-His-FluA-MiddleLinker-Fok(inactive)3<br /> |
- | - XBL pEXHisFluASplitFokIRBSStrepDigSplitFokA<br> | + | - pMA-His-FluA-ShortLinker-Fok(inactive)1<br /> |
- | - BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA<br> | + | - pMA-His-FluA-SplitLinker-Fok(inactive)2<br /> |
- | <br>*Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each | + | - pMA-Dbsa 2</small><br /> |
- | - induced with 0,7mM IPTG and took samples T0-T5<br> | + | <br /> |
- | - centrifuged in buckets 17', 4000rpm, 4°C<br> | + | </div> |
- | - eluted in 20 ml TES in each bucket<br> | + | <ul style="margin-left: 40px;"> |
| + | <li> Transformation of</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- XBL |
| + | pEXHisFluASplitFokIRBSStrepDigSplitFokA<br /> |
| + | - BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l |
| + | flasks in 600ml DYT medium each |
| + | - induced with 0,7mM IPTG and took samples T0-T5</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- centrifuged |
| + | in buckets |
| + | 17', 4000rpm, 4°C<br /> |
| + | - eluted in 20 ml TES in each bucket<br /> |
| .... | | .... |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>PET 39 b+ ssDNA "PCR" with new template from digestion of |
| + | yesterday -> very few product, as well as non specific ones |
| + | </li> |
| + | <li>PET 39 b+ ssDNA "PCR" with more cycles and higher annealing |
| + | temperature |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;">06.10.09, Laura, Manu, |
| + | Julia, Christophe, Caro, Hannes, |
| + | Gerrit, Anika</h3> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>digest |
| + | - pBAD vector </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>DNA: |
| + | 16.8µl<br /> |
| + | enzyme: 1µl AgeI<br /> |
| + | buffer: 2µl buffer 1 <br /> |
| + | - pBAD insert <br /> |
| + | DNA: 16.8µl<br /> |
| + | enzyme: 1µl XmaI<br /> |
| + | buffer: 2µl buffer 4 <br /> |
| + | BSA (10X): 2µl <br /> |
| + | - pJS 418/419 <br /> |
| + | DNA: 10µl<br /> |
| + | enzyme: 1µl PstI and 1.5 µl Xba/<br /> |
| + | buffer: 3µl buffer 3 <br /> |
| + | BSA (10X): 3µl <br /> |
| + | - pExStrepDigSplitFoka/pExHisDigSplitFoka <br /> |
| + | DNA: 16.8µl<br /> |
| + | enzyme: 1µl PstI and 1.5 µl Xba/<br /> |
| + | buffer: 3µl buffer 3 <br /> |
| + | BSA (10X): 3µl </small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA: |
| + | 5µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>enzyme: |
| + | 1µl PstI |
| + | and 1.5 µl Xba/<br /> |
| + | buffer: 1µl buffer 3 <br /> |
| + | BSA (10X): 1µl </small><br /> |
| + | <br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>preparative gels of digests |
| + | </li> |
| + | </ul> |
| + | <div style="margin-left: 80px;"></div> |
| + | <table style="text-align: left; width: 750px; margin-left: 40px;" |
| + | border="1" cellpadding="0" cellspacing="1"> |
| + | <tbody> |
| + | <tr> |
| + | <td><img class="art-article" |
| + | style="width: 350px; height: 280px;" alt="" |
| + | src="https://static.igem.org/mediawiki/2009/d/d5/Freiburg09_061009_bad_js.JPG" /></td> |
| + | <td><img class="art-article" |
| + | style="width: 423px; height: 280px;" alt="" |
| + | src="https://static.igem.org/mediawiki/2009/7/75/Freiburg09_061009_js_foka.JPG" /></td> |
| + | </tr> |
| + | <tr> |
| + | <td><small>preparative agarose gel, lanes: 1. |
| + | Genruler DNA ladder |
| + | mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418</small></td> |
| + | <td><small>preparative agarose gel, lanes: 1. |
| + | Genruler DNA ladder |
| + | mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <div style="margin-left: 40px;"><small><br /> |
| + | interpretation: |
| + | pExStrepDigSplitFoka seemed |
| + | to be a wrong construct, the other constructs showed bands of the right |
| + | size even if the concentration of the PCR pBAD insert seems to be very |
| + | low |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>testdigest of pMAdsba |
| + | DNA: 5µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>enzyme: |
| + | 1µl PstI |
| + | and 1.5 µl Xba/<br /> |
| + | buffer: 1µl buffer 3 <br /> |
| + | BSA (10X): 1µl </small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>His-tag purification of HisDigSplitFoka (periplasm export) |
| + | with Ni-NTA |
| + | column. Washing buffer: 25mM imidazole |
| + | -cells were sonicated for 2 x 1min before filtering with |
| + | 0.45µm and 0.22µm filter |
| + | </li> |
| + | </ul> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Ligation |
| + | - Dephphorylation of pBAD Gelex</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | 1µl Fast AP<br /> |
| + | - 5.5µl fast AP buffer<br /> |
| + | -1.5µl water<br /> |
| + | --> Solution was given to eluat |
| + | - for each ligation:<br /> |
| + | - 6µl Insert<br /> |
| + | -3µl Vetor<br /> |
| + | -1µl Quickligase<br /> |
| + | -10µl buffer<br /> |
| + | --> pBAD </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Insert (Dummy)</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>--> |
| + | pJS419+HisDigSplitFoka |
| + | -->pJ418+HisDigSplitFoka |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Transformation |
| + | --> pBAD </li> |
| + | <li> Insert (Dummy)</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>--> |
| + | pJS419+HisDigSplitFoka |
| + | -->pJ418+HisDigSplitFoka |
| + | - in XLblue<br /> |
| + | -pBAD was plated on AMP plates<br /> |
| + | - Both pJS... were plated on CM plates</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li><big>Ligation and Transformation of</big></li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"> <small>Ligation:per |
| + | sample 8µl H2O; 3µl vector; |
| + | 6µl insert; 2µl Quick Ligase buffer; 1µl |
| + | Quick Ligase |
| + | Transformation:<b>Defrost competent cells on ice:</b>(100 |
| + | µl); |
| + | <b>add of the ligation:</b> 5 µl; |
| + | <b>DNA and cells:</b> mix softly by knocking; <b>Incubation |
| + | on ice for:</b> 20-30 min; |
| + | <b>Heat shock at:</b> 42°C for 40 sec; |
| + | <b>Cool off on ice for:</b> 5 min; |
| + | <b>add sterile LB(or dyt)medium:</b> 900 µl; <b>Incubation |
| + | in(shaker)at:</b> 37°C for 60-70 min; <b>Plate |
| + | cells on LB+antibiotic plates:</b> ampicillin; |
| + | <b>ligation:</b> 2 plates: 1. 50µl cells <b>2. |
| + | centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the |
| + | restcells and plate out:</b> -pMA HisDig Middle Linker Foki<br /> |
| + | <br /> |
| + | -pMA CAT<br /> |
| + | -pEX Strep FluA SL FokA<br /> |
| + | -pEX Strep FluA LL Foki<br /> |
| + | -pEX Strep FluA ML Foki<br /> |
| + | -pEX Strep FluA ML Foka<br /> |
| + | -pEX Strep FluA LL Foka</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Sequencing: |
| + | 27µl H2O; 3µl DNA |
| + | -pMA Dbsa clone 2</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-pMA |
| + | HisDigSplitFokA clone |
| + | 2<br /> |
| + | -pMA HisFluASL Foki clone 1<br /> |
| + | -pMA HisFluASplitFoki clone 2<br /> |
| + | -pMA StrepDigLLFoki clone 3<br /> |
| + | -pMA HisFluaMLFoki clone 3<br /> |
| + | -pMA StrepDigSplitFokA clone 1<br /> |
| + | -pMA StrepDigMLFoki clone 1<br /> |
| + | -pMA StrepDigSLFoki clone 2<br /> |
| + | -pMA HisDigLLFoki clone 1<br /> |
| + | -pMA StrepDigSplitFoki clone 3<br /> |
| + | -pMA HisDigSplitFoki clone 1<br /> |
| + | -pMA HisFluaLLFoki clone 3</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Inoculation |
| + | of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room |
| + | overnight</li> |
| + | <li>Yesterdays improved pcr resulted in a lot of product, but |
| + | still |
| + | unspecific ones.. made new one with even higher annealing temperature |
| + | (69°C) and a bit less cycles (35) |
| + | </li> |
| + | <li>New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1 |
| + | and A4 _see |
| + | gele_ |
| + | no differend results were obtained compaired to the assays using |
| + | unphorphorylated oligos |
| + | </li> |
| + | <li>Started dialysis to transfer the leftover AGO-proteins into |
| + | the assay |
| + | buffer |
| + | </li> |
| + | </ul> |
| + | <small> |
| + | FokI wildtype cutting<br> |
| | | |
- | <br>*PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -> very few product, as well as non specific ones | + | Hybridize M13 ssDNA with Fok control oligos 2 and 3 |
- | <br>*PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature | + | <table border="1"> |
| + | <tr> |
| + | <td align="center"> Volume </td> |
| + | <td>Reagent</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">10 µl</td> |
| + | <td>M13 ssDNA (c = 127 ng/µl, 17.08.09, sample #5.2)</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">5 µl</td> |
| + | <td>MgCl2 (5 mM)</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">5 µl</td> |
| + | <td>Tris-HCl pH 8 (100 mM)</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">2 µl</td> |
| + | <td>Fok control 2 (10 ng/µl; 1 µM)</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">2 µl</td> |
| + | <td>Fok control 3 (10 ng/µl; 1 µM)</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">30 µl</td> |
| + | <td></td> |
| + | </tr> |
| + | </table><br> |
| + | The hybridization was also done with just Fok control 2 and with no oligos at all.<br> |
| + | Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).<br> |
| | | |
- | <h3>06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika</h3>
| + | 10 µl of the hybridized DNA were used for a cutting experiment with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI (wildtype Fok from NEB). The digest was incubated for 30 minutes at 37°C.<br> |
- | <br>*digest
| + | |
- | - pBAD vector <br>
| + | |
- | DNA: 16.8µl<br>
| + | |
- | enzyme: 1µl AgeI<br>
| + | |
- | buffer: 2µl buffer 1 <br>
| + | |
| | | |
- | - pBAD insert <br>
| |
- | DNA: 16.8µl<br>
| |
- | enzyme: 1µl XmaI<br>
| |
- | buffer: 2µl buffer 4 <br>
| |
- | BSA (10X): 2µl <br>
| |
| | | |
- | - pJS 418/419 <br>
| + | <table> |
- | DNA: 10µl<br>
| + | <tr> |
- | enzyme: 1µl PstI and 1.5 µl Xba/<br>
| + | |
- | buffer: 3µl buffer 3 <br>
| + | |
- | BSA (10X): 3µl <br>
| + | |
- | | + | |
- | - pExStrepDigSplitFoka/pExHisDigSplitFoka <br>
| + | |
- | DNA: 16.8µl<br>
| + | |
- | enzyme: 1µl PstI and 1.5 µl Xba/<br>
| + | |
- | buffer: 3µl buffer 3 <br>
| + | |
- | BSA (10X): 3µl <br>
| + | |
- | | + | |
- | <br>*testdigest of pExHisFluASplitFoki_StrepDigSplitFoka
| + | |
- | DNA: 5µl<br>
| + | |
- | enzyme: 1µl PstI and 1.5 µl Xba/<br>
| + | |
- | buffer: 1µl buffer 3 <br>
| + | |
- | BSA (10X): 1µl <br>
| + | |
- | <br>*preparative gels of digests
| + | |
- | <table border="0"> | + | |
- | <tr><td> | + | |
- | [[Image:Freiguburg09_061009_bad_js.JPG|none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418|300x300px]]
| + | |
- | </td>
| + | |
| <td> | | <td> |
- | [[Image:Freiburg09_061009_js_foka.JPG |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
| + | <img src="https://static.igem.org/mediawiki/2009/5/5a/Freiburg09_061009_fokI_cutting2_crop.JPG" name="Agarose gel of FokI cutting M13 ssDNA" width="188" height="230" /><br> |
- | </td></tr></table>
| + | Agarose gel of FokI cutting M13 ssDNA |
- | interpretation: pExStrepDigSplitFoka seemed to be a wrong construct, the other constructs showed bands of the right size even if the concentration of the PCR pBAD insert seems to be very low
| + | </td> |
- | <br>*testdigest of pMAdsba | + | |
- | DNA: 5µl<br>
| + | |
- | enzyme: 1µl PstI and 1.5 µl Xba/<br>
| + | |
- | buffer: 1µl buffer 3 <br>
| + | |
- | BSA (10X): 1µl <br>
| + | |
| | | |
- | <br>*His-tag purification of HisDigSplitFoka (periplasm export) with Ni-NTA column. Washing buffer: 25mM imidazole | + | <td> |
- | -cells were sonicated for 2 x 1min before filtering with 0.45µm and 0.22µm filter
| + | <img src="https://static.igem.org/mediawiki/2009/4/4f/Freiburg09_061009_fokI_cutting3_crop.jpg" name="Agarose gel of FokI cutting M13 ssDNA"width="188" height="230" /><br> |
- | | + | Agarose gel of FokI cutting M13 ssDNA |
- | <br>*Ligation | + | </td> |
- | - Dephphorylation of pBAD Gelex<br>
| + | </tr> |
- | - 1µl Fast AP<br>
| + | </table> |
- | - 5.5µl fast AP buffer<br>
| + | |
- | -1.5µl water<br>
| + | |
- | | + | |
- | --> Solution was given to eluat
| + | |
| | | |
- | - for each ligation:<br>
| + | Lane 1: GeneRuler DNA Ladder Mix (Fermentas, 6 µl); <br> |
- | - 6µl Insert<br>
| + | Lane 2: Untreated M13 ssDNA (250 ng); <br> |
- | -3µl Vetor<br>
| + | Lane 3: Digest of M13 ssDNA with FokI and without oligo; <br> |
- | -1µl Quickligase<br>
| + | Lane 4: Digest of M13 ssDNA with FokI and with one oligo; <br> |
- | -10µl buffer<br>
| + | Lane 5: Digest of M13 ssDNA with FokI and two oligos.</small><br><br> |
| | | |
- | --> pBAD <br>* Insert (Dummy)<br>
| |
- | --> pJS419+HisDigSplitFoka
| |
- | -->pJ418+HisDigSplitFoka
| |
- | <br>* Transformation
| |
| | | |
- | --> pBAD <br>* Insert (Dummy)<br>
| |
- | --> pJS419+HisDigSplitFoka
| |
- | -->pJ418+HisDigSplitFoka
| |
| | | |
- | - in XLblue<br>
| |
| | | |
- | -pBAD was plated on AMP plates<br>
| + | <h3 style="margin-left: 40px;">07.10.09 Laura,Christoph, |
- | - Both pJS... were plated on CM plates<br> | + | Hannes, Timo, Julia, Caro, Anika</h3> |
- | | + | <div style="margin-left: 40px;"><br /> |
- | <br>*'''Ligation and Transformation of'''
| + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | | + | <li>SDS gel of protein purification of HisDigSplitFoka |
- | Ligation:per sample 8µl H2O; 3µl vector; 6µl insert; 2µl Quick Ligase buffer; 1µl Quick Ligase
| + | (periplasm) |
- | | + | from 06.10.09 |
- | | + | [[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS |
- | Transformation:<b>Defrost competent cells on ice:</b>(100 µl);
| + | gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker |
- | <b>add of the ligation:</b> 5 µl;
| + | broad range, Elution fraction 1, Elution fraction 2, Elution fraction |
- | <b>DNA and cells:</b> mix softly by knocking;
| + | 3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution |
- | <b>Incubation on ice for:</b> 20-30 min;
| + | fraction 6, flow through fraction 3, washing fraction 2, periplasm |
- | <b>Heat shock at:</b> 42°C for 40 sec;
| + | extract (frozen over night at -80°C)|400x400px]] |
- | <b>Cool off on ice for:</b> 5 min;
| + | </li> |
- | <b>add sterile LB(or dyt)medium:</b> 900 µl;
| + | </ul> |
- | <b>Incubation in(shaker)at:</b> 37°C for 60-70 min;
| + | <ul style="margin-left: 40px;"> |
- | <b>Plate cells on LB+antibiotic plates:</b> ampicillin;
| + | <li> Inoculation 4 Clones respectively:</li> |
- | <b>ligation:</b> 2 plates: 1. 50µl cells
| + | </ul> |
- | <b>2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the restcells and plate out:</b>
| + | <div style="margin-left: 40px;"><small>- |
- | | + | pMA-His-Dig-MiddleLinker-Foki in XLBlue<br /> |
- | -pMA HisDig Middle Linker Foki<br>
| + | - pJS419-HIs-Dig-Split-Foka- in XLBlue<br /> |
- | -pMA CAT<br> | + | - pJS418-HIs-DIg-Split-Foka- in XLBlue<br /> |
- | -pEX Strep FluA SL FokA<br>
| + | - pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest<br /> |
- | -pEX Strep FluA LL Foki<br>
| + | - pEX-Strep-FluA-LongLinker-Foki in XL1blue rest<br /> |
- | -pEX Strep FluA ML Foki<br>
| + | - pMA-CAT in XLblue 10µl<br /> |
- | -pEX Strep FluA ML Foka<br>
| + | -pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest<br /> |
- | -pEX Strep FluA LL Foka<br>
| + | 2 Clones respectively:<br /> |
- | | + | |
- | | + | |
- | <br>*Sequencing:
| + | |
- | | + | |
- | 27µl H2O; 3µl DNA
| + | |
- | | + | |
- | -pMA Dbsa clone 2<br>
| + | |
- | -pMA HisDigSplitFokA clone 2<br>
| + | |
- | -pMA HisFluASL Foki clone 1<br>
| + | |
- | -pMA HisFluASplitFoki clone 2<br>
| + | |
- | -pMA StrepDigLLFoki clone 3<br> | + | |
- | -pMA HisFluaMLFoki clone 3<br>
| + | |
- | -pMA StrepDigSplitFokA clone 1<br>
| + | |
- | -pMA StrepDigMLFoki clone 1<br>
| + | |
- | -pMA StrepDigSLFoki clone 2<br>
| + | |
- | -pMA HisDigLLFoki clone 1<br>
| + | |
- | -pMA StrepDigSplitFoki clone 3<br>
| + | |
- | -pMA HisDigSplitFoki clone 1<br>
| + | |
- | -pMA HisFluaLLFoki clone 3<br>
| + | |
- | | + | |
- | <br>*Inoculation
| + | |
- | | + | |
- | of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room overnight<br>
| + | |
- | | + | |
- | <br>*Yesterdays improved pcr resulted in a lot of product, but still unspecific ones.. made new one with even higher annealing temperature (69°C) and a bit less cycles (35) | + | |
- | | + | |
- | <br>*New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1 and A4 _see gele_
| + | |
- | no differend results were obtained compaired to the assays using unphorphorylated oligos
| + | |
- | | + | |
- | <br>*Started dialysis to transfer the leftover AGO-proteins into the assay buffer
| + | |
- | | + | |
- | <h3>07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika</h3>
| + | |
- | <br>*SDS gel of protein purification of HisDigSplitFoka (periplasm) from 06.10.09
| + | |
- | | + | |
- | | + | |
- | [[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker broad range, Elution fraction 1, Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution fraction 6, flow through fraction 3, washing fraction 2, periplasm extract (frozen over night at -80°C)|400x400px]] | + | |
- | | + | |
- | | + | |
- | <br>* Inoculation | + | |
- | 4 Clones respectively:<br> | + | |
- | - pMA-His-Dig-MiddleLinker-Foki in XLBlue<br> | + | |
- | - pJS419-HIs-Dig-Split-Foka- in XLBlue<br> | + | |
- | - pJS418-HIs-DIg-Split-Foka- in XLBlue<br> | + | |
- | - pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest<br> | + | |
- | - pEX-Strep-FluA-LongLinker-Foki in XL1blue rest<br> | + | |
- | - pMA-CAT in XLblue 10µl<br> | + | |
- | -pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest<br> | + | |
- | | + | |
- | 2 Clones respectively:<br> | + | |
| -pEX-His-FluA-Split-Foki + Glycerolstock vom 26.08.09 | | -pEX-His-FluA-Split-Foki + Glycerolstock vom 26.08.09 |
| - pEX-Strep-Dig-Split-Foka + Glycerolstock vom 29.08.09 | | - pEX-Strep-Dig-Split-Foka + Glycerolstock vom 29.08.09 |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Starter culture of pEx-DsbAHisDigSplitFoka in |
| + | Bl21de3 |
| + | </li> |
| + | <li>two step PCR assembly of DsbA, His_Fos, and SplitFoka |
| + | - program name: Assembly</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- three |
| + | different samples: |
| + | 1. without DMSO, 2. with DMSO, 3. without |
| + | DMSO and with last primers just added after first step |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>preparative gel of the PCR samples |
| + | -> primer haven't been diluted an probably made all secondary |
| + | structures, has to be repeated |
| + | </li> |
| + | <li>digest of pBAD with AgeI and new PCR with insert digested |
| + | with XmaI |
| + | </li> |
| + | <li>preparative gel with digest and </li> |
| + | <li>test digest of pJS419_HisDigSplitFoka and |
| + | pJS419_StrepDigSplitFoka |
| + | DNA: 5µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>Enzymes: |
| + | 0.5µl |
| + | of BamHI and MfeI each<br /> |
| + | Buffer: 1µl buffer 4<br /> |
| + | BSA (10fold): 1µl<br /> |
| + | Water: 2µl<br /> |
| + | -> pJS419_HisDigSplitFoka showed bands of the right size and was |
| + | sequenced with primer sf_lac P1</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>plasmidpreparation of - pEXHisFluASplitFoki</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | pExStrepDigSplitFoka<br /> |
| + | -> low concentrations</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and |
| + | RBS_StrepDigSplitFoka |
| + | pExHisFluASplitFoki: 15µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>Enzymes: |
| + | 1µl of |
| + | PstI and 1.5µl SpeI<br /> |
| + | Buffer: 3µl buffer 2<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 9µl<br /> |
| + | pExStrepDigSplitFoka: 15µl<br /> |
| + | Enzymes: 1µl of XbaI and 1.5µl PstI<br /> |
| + | Buffer: 3µl buffer 3<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 9µl<br /> |
| + | pExStrepDigSplitFoka: 10µl<br /> |
| + | Enzymes: 1µl of XbaI and 1.5µl PstI<br /> |
| + | Buffer: 3µl buffer 3<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 14µl</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Gelextraction of digest |
| + | 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>2)Vector: |
| + | pEX-His-Flua-Split-Foki (digested with spe, pst)<br /> |
| + | 3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>preparative gels of digests |
| + | </li> |
| + | </ul> |
| + | <table style="text-align: left; width: 750px; margin-left: 40px;" border="1" |
| + | cellpadding="2" cellspacing="0"> |
| + | <tbody> |
| + | <tr> |
| + | <td><img style="width: 400px; height: 303px;" |
| + | alt="" |
| + | src="https://static.igem.org/mediawiki/2009/0/0a/Freiburg09_7.10_digestcaro.JPG"></td> |
| + | <td><img style="width: 400px; height: 303px;" |
| + | alt="" |
| + | src="https://static.igem.org/mediawiki/2009/f/f3/Freiburg09_7.10_digest2_caro.JPG"></td> |
| + | </tr> |
| + | <tr> |
| + | <td><small>preparative agarose gel, lanes: 1. |
| + | Insert2. pEX-Vector, 3. PCR- RBS Product</small></td> |
| + | <td><small>preparative agarose gel, lanes: 1. |
| + | Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. |
| + | pExStrepDigSplitFoka</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| | | |
- | | + | <div style="margin-left: 40px;"><small>RBS PCR |
- | <br>* Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3 | + | Product: m= 90 mg<br /> |
- | <br>*two step PCR assembly of DsbA, His_Fos, and SplitFoka
| + | vector: 0 0 130 mg</small><br /> |
- | - program name: Assembly<br>
| + | <br /> |
- | - three different samples: 1. without DMSO, 2. with DMSO, 3. without DMSO and with last primers just added after first step
| + | </div> |
- | <br>*preparative gel of the PCR samples | + | <ul style="margin-left: 40px;"> |
- | -> primer haven't been diluted an probably made all secondary structures, has to be repeated
| + | <li> Ligation |
- | <br>*digest of pBAD with AgeI and new PCR with insert digested with XmaI
| + | - 6µl Insert</li> |
- | <br>*preparative gel with digest and
| + | </ul> |
- | <br>*test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka
| + | <div style="margin-left: 40px;"><small>-3µl |
- | DNA: 5µl<br>
| + | Vetor<br /> |
- | Enzymes: 0.5µl of BamHI and MfeI each<br>
| + | -1µl Quickligase<br /> |
- | Buffer: 1µl buffer 4<br>
| + | -10µl buffer<br /> |
- | BSA (10fold): 1µl<br>
| + | pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br /> |
- | Water: 2µl<br>
| + | </small><br /> |
- | -> pJS419_HisDigSplitFoka showed bands of the right size and was sequenced with primer sf_lac P1<br>
| + | </div> |
- | <br>*plasmidpreparation of
| + | <ul style="margin-left: 40px;"> |
- | - pEXHisFluASplitFoki<br> | + | <li>Transformation |
- | - pExStrepDigSplitFoka<br>
| + | - All in XL1blue</li> |
- | -> low concentrations<br> | + | </ul> |
- | <br>*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka | + | <div style="margin-left: 40px;"><small>- Vector: |
- | pExHisFluASplitFoki: 15µl<br>
| + | pEX-strep-Duig-Split-Foka<br /> |
- | Enzymes: 1µl of PstI and 1.5µl SpeI<br>
| + | - Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka</small><br /> |
- | Buffer: 3µl buffer 2<br>
| + | <br /> |
- | BSA (100fold): 0.5µl<br>
| + | </div> |
- | Water: 9µl<br>
| + | <ul style="margin-left: 40px;"> |
- | | + | <li>Stardet production of Phages baering the phagmid vektors |
- | pExStrepDigSplitFoka: 15µl<br>
| + | with |
- | Enzymes: 1µl of XbaI and 1.5µl PstI<br>
| + | pJS_TorA-flag-AGO-noAmber-CDg3p and the pJS_TorA-flag-AGO-CDg3p, 480 ml |
- | Buffer: 3µl buffer 3<br>
| + | each. see phageproduction protocoll day 1 |
- | BSA (100fold): 0.5µl<br>
| + | </li> |
- | Water: 9µl<br>
| + | <li>prepaired ELISA with anti-flag antibodies |
- | | + | </li> |
- | pExStrepDigSplitFoka: 10µl<br>
| + | <li>made electro competent cells for transformation with the |
- | Enzymes: 1µl of XbaI and 1.5µl PstI<br>
| + | ago |
- | Buffer: 3µl buffer 3<br>
| + | phagmidbibliothek |
- | BSA (100fold): 0.5µl<br>
| + | </li> |
- | Water: 14µl<br>
| + | </ul> |
- | <br>* Gelextraction of digest
| + | <h3 style="margin-left: 40px;">08.10.09 Manu, Christoph, |
- | 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) <br>
| + | Julia, Caro, Timo, Hannes </h3> |
- | 2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br>
| + | <div style="margin-left: 40px;"><br /> |
- | 3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br>
| + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | | + | <li>Poured SDS-Gels</li> |
- | <br>*preparative gels of digests
| + | <li>Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet |
- | <table border="0">
| + | dilute on |
- | <tr><td>
| + | OD600=0,4 in 50ml LB+Tet</li> |
- | [[Image:|none|thumb|preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product]]
| + | </ul> |
- | </td>
| + | <div style="margin-left: 40px;"><small> |
- | <td>
| + | -Transfomation with 3µl M13 Phage stock (-80°) at |
- | [[Image: |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
| + | 10:30Uhr , incubated for 4-5 hours at 37°C in shaker<br /> |
- | </td></tr></table> | + | -Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C<br /> |
- | | + | -Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml |
- | RBS PCR Product: m= 90 mg<br>
| + | precipitation over night on ice in -4°C room</small><br /> |
- | vector: 0 0 130 mg<br>
| + | <br /> |
- | | + | </div> |
- | <br>* Ligation | + | <ul style="margin-left: 40px;"> |
- | - 6µl Insert<br> | + | <li>Miniprep |
- | -3µl Vetor<br>
| + | - 2 clones respectively:</li> |
- | -1µl Quickligase<br>
| + | </ul> |
- | -10µl buffer<br> | + | <div style="margin-left: 40px;"><small>-A = pEX |
- | | + | strep dig split |
- | pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br>
| + | foka |
- | <br>*Transformation
| + | </small><br /> |
- | | + | </div> |
- | - All in XL1blue<br>
| + | <ul style="margin-left: 40px;"> |
- | - Vector: pEX-strep-Duig-Split-Foka<br> | + | <li>Finished Phage production(see protocol day 2): We obtained |
- | - Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br> | + | approximately 3.8</li> |
- | | + | <li>10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1</li> |
- | <br>*Stardet production of Phages baering the phagmid vektors with pJS_TorA-flag-AGO-noAmber-CDg3p and the pJS_TorA-flag-AGO-CDg3p, 480 ml each. see phageproduction protocoll day 1 | + | <li>10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of |
- | <br>*prepaired ELISA with anti-flag antibodies | + | them to |
- | <br>*made electro competent cells for transformation with the ago phagmidbibliothek | + | the anti-Flag ELISA</li> |
- | | + | </ul> |
- | <h3>08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes </h3> | + | <div style="margin-left: 40px;"><br /> |
- | <br>*Poured SDS-Gels<br> | + | </div> |
- | <br>*Phage ss DNA | + | <ul style="margin-left: 40px;"> |
- | | + | <li>Anti-Flag ELISA was successfull with a slight but detctable |
- | -Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet<br> | + | signal via |
- | -Transfomation with 3µl M13 Phage stock (-80°) at 10:30Uhr , incubated for 4-5 hours at 37°C in shaker<br> | + | anti M13 VCS Antibodies (with peroxidase):</li> |
- | -Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C<br> | + | </ul> |
- | -Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml precipitation over night on ice in -4°C room<br> | + | <div style="margin-left: 40px;"><small>see |
- | | + | 405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to |
- | <br>*Miniprep | + | positive control (G5-6) and negative control (G3-4 and D3-10) detected |
- | | + | about half an hour after the ABTS substrat was addet. |
- | - 2 clones respectively:<br> | + | <br /> |
- | -A = pEX strep dig split foka | + | <br /> |
- | | + | </small></div> |
- | <br>*Finished Phage production(see protocol day 2): We obtained approximately 3.8<br>*10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1<br>*10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of them to the anti-Flag ELISA<br> | + | <table style="margin-left: 40px;" id="table_1" |
- | | + | border="2" cellpadding="2" cellspacing="0"> |
- | <br>*Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):<br> | + | <small></small><tbody> |
- | see 405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to positive control (G5-6) and negative control (G3-4 and D3-10) detected about half an hour after the ABTS substrat was addet. | + | <tr> |
- | | + | <small></small><td><small> </small></td> |
- | <> 1 2 3 4 5 6 7 8 9 10 11 12 <br> | + | <small></small><td><small>1</small></td> |
- | A 0.0470 0.0420 0.0440 0.0430 0.0480 0.0510 0.0460 0.0470 0.0470 0.0480 0.0450 0.0460<br> | + | <small></small><td><small>2</small></td> |
- | B 0.0470 0.0460 0.0480 0.0480 0.0460 0.0450 0.0470 0.0560 0.0470 0.0450 0.0450 0.0440<br> | + | <small></small><td><small>3</small></td> |
- | C 0.0460 0.0470 0.0500 0.0450 0.0430 0.0460 0.0450 0.0460 0.0430 0.0420 0.0440 0.0470<br> | + | <small></small><td><small>4</small></td> |
- | D 0.0490 0.0470 0.1590 0.1610 0.1920 0.1720 0.2210 0.2500 0.2130 0.2680 0.0460 0.0480<br> | + | <small></small><td><small>5</small></td> |
- | E 0.0490 0.0490 0.0450 0.0490 0.0430 0.0490 0.0460 0.0430 0.0460 0.0420 0.0450 0.0440<br> | + | <small></small><td><small>6</small></td> |
- | F 0.0490 0.0430 0.0470 0.0440 0.0470 0.0470 0.0440 0.0450 0.0500 0.0460 0.0450 0.0450<br> | + | <small></small><td><small>7</small></td> |
- | G 0.0500 0.0440 0.2530 0.2510 3.9180 3.8800 0.9560 0.9380 1.2420 1.2360 0.0440 0.0460<br> | + | <small></small><td><small>8</small></td> |
- | H 0.0480 0.0430 0.0510 0.0470 0.0480 0.0460 0.0470 0.0450 0.0450 0.0440 0.0460 0.0460<br> | + | <small></small><td><small>9</small></td> |
- | | + | <small></small><td><small>10</small></td> |
- | <br>* Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction | + | <small></small><td><small>11</small></td> |
- | | + | <small></small><td><small>12</small></td> |
- | <br>*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka | + | <small></small></tr> |
- | pExHisFluASplitFoki: 15µl<br> | + | <small></small><tr> |
- | Enzymes: 1µl of PstI and 1.5µl SpeI<br> | + | <small></small><td><small>A</small></td> |
- | Buffer: 3µl buffer 2<br> | + | <small></small><td><small>0.0470</small></td> |
- | BSA (100fold): 0.5µl<br> | + | <small></small><td><small>0.0420</small></td> |
- | Water: 9µl<br> | + | <small></small><td><small>0.0440</small></td> |
- | | + | <small></small><td><small>0.0430</small></td> |
- | pExHisDigSplitFoka: 15µl<br> | + | <small></small><td><small>0.0480</small></td> |
- | Enzymes: 1µl of XbaI and 1.5µl PstI<br> | + | <small></small><td><small>0.0510</small></td> |
- | Buffer: 3µl buffer 3<br> | + | <small></small><td><small>0.0460</small></td> |
- | BSA (100fold): 0.5µl<br> | + | <small></small><td><small>0.0470</small></td> |
- | Water: 14µl<br> | + | <small></small><td><small>0.0470</small></td> |
- | | + | <small></small><td><small>0.0480</small></td> |
- | RBSStrepDigSplitFoka: 5µl<br> | + | <small></small><td><small>0.0450</small></td> |
- | Enzymes: 1µl of XbaI and 1.5µl PstI<br> | + | <small></small><td><small>0.0460</small></td> |
- | Buffer: 3µl buffer 3<br> | + | <small></small></tr> |
- | BSA (100fold): 0.5µl<br> | + | <small></small><tr> |
- | Water: 19µl<br> | + | <small></small><td><small>B</small></td> |
- | ->made two digest of PCR construct<br> | + | <small></small><td><small>0.0470</small></td> |
- | <br>* Gelextraction of digest | + | <small></small><td><small>0.0460</small></td> |
- | 1) Insert: His-Dig-Split-Foka( digested with xba and pst) <br> | + | <small></small><td><small>0.0480</small></td> |
- | 2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br> | + | <small></small><td><small>0.0480</small></td> |
- | 3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br> | + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0560</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small></tr> |
| + | <small></small><tr> |
| + | <small></small><td><small>C</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0500</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0430</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0430</small></td> |
| + | <small></small><td><small>0.0420</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small></tr> |
| + | <small></small><tr> |
| + | <small></small><td><small>D</small></td> |
| + | <small></small><td><small>0.0490</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.1590</small></td> |
| + | <small></small><td><small>0.1610</small></td> |
| + | <small></small><td><small>0.1920</small></td> |
| + | <small></small><td><small>0.1720</small></td> |
| + | <small></small><td><small>0.2210</small></td> |
| + | <small></small><td><small>0.2500</small></td> |
| + | <small></small><td><small>0.2130</small></td> |
| + | <small></small><td><small>0.2680</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0480</small></td> |
| + | <small></small></tr> |
| + | <small></small><tr> |
| + | <small></small><td><small>E</small></td> |
| + | <small></small><td><small>0.0490</small></td> |
| + | <small></small><td><small>0.0490</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0490</small></td> |
| + | <small></small><td><small>0.0430</small></td> |
| + | <small></small><td><small>0.0490</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0430</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0420</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small></tr> |
| + | <small></small><tr> |
| + | <small></small><td><small>F</small></td> |
| + | <small></small><td><small>0.0490</small></td> |
| + | <small></small><td><small>0.0430</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0500</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small></tr> |
| + | <small></small><tr> |
| + | <small></small><td><small>G</small></td> |
| + | <small></small><td><small>0.0500</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small><td><small>0.2530</small></td> |
| + | <small></small><td><small>0.2510</small></td> |
| + | <small></small><td><small>3.9180</small></td> |
| + | <small></small><td><small>3.8800</small></td> |
| + | <small></small><td><small>0.9560</small></td> |
| + | <small></small><td><small>0.9380</small></td> |
| + | <small></small><td><small>1.2420</small></td> |
| + | <small></small><td><small>1.2360</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small></tr> |
| + | <small></small><tr> |
| + | <small></small><td><small>H</small></td> |
| + | <small></small><td><small>0.0480</small></td> |
| + | <small></small><td><small>0.0430</small></td> |
| + | <small></small><td><small>0.0510</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0480</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0470</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0450</small></td> |
| + | <small></small><td><small>0.0440</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small><td><small>0.0460</small></td> |
| + | <small></small></tr> |
| + | <small></small> |
| + | </tbody> |
| + | </table> |
| + | <div style="margin-left: 40px;"> |
| + | <br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Made new PCR for ssDNA from pET39b+ fragment and |
| + | gained approximately |
| + | 200 ng of ssDNA after PCR and gelextraction |
| + | </li> |
| + | <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and |
| + | RBS_StrepDigSplitFoka |
| + | pExHisFluASplitFoki: 15µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>Enzymes: |
| + | 1µl of PstI and 1.5µl SpeI<br /> |
| + | Buffer: 3µl buffer 2<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 9µl<br /> |
| + | pExHisDigSplitFoka: 15µl<br /> |
| + | Enzymes: 1µl of XbaI and 1.5µl PstI<br /> |
| + | Buffer: 3µl buffer 3<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 14µl<br /> |
| + | RBSStrepDigSplitFoka: 5µl<br /> |
| + | Enzymes: 1µl of XbaI and 1.5µl PstI<br /> |
| + | Buffer: 3µl buffer 3<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 19µl<br /> |
| + | ->made two digest of PCR construct<br /> |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Gelextraction of digest |
| + | 1) Insert: His-Dig-Split-Foka( digested with xba and pst) </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>2)Vector: |
| + | pEX-His-Flua-Split-Foki (digested with spe, pst)<br /> |
| + | 3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br /> |
| and also PCR purification of RBS-Strep-Dig-Split-FokA | | and also PCR purification of RBS-Strep-Dig-Split-FokA |
- | <br>*preparative gels of digests | + | </small><br /> |
- | ->see picture<br> | + | </div> |
- | <br>*ligation of | + | <ul style="margin-left: 40px;"> |
- | -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka<br> | + | <li>preparative gels of digests |
- | -pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from gelex)<br> | + | ->see picture</li> |
- | -pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR purification)<br> | + | </ul> |
- | <br>*transformation of ligations | + | <div style="margin-left: 40px;"><br /> |
- | <br>*inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol | + | </div> |
- | <br>*new two step PCR with all three plasmids for Fos construct (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution and preparative agarose gel | + | <ul style="margin-left: 40px;"> |
- | ->interpretation: no band of 921bp was visible<br> | + | <li>ligation of |
- | <br>*one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel | + | -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka</li> |
- | ->see picture<br> | + | </ul> |
- | ->interpretation: pMAFos and pMASplitFoka showed bands of the right size (230bp and 657bp respectively), of pMADsbA no product was visible<br> | + | <div style="margin-left: 40px;"><small>-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka |
- | <br>*new one step PCR of pMADsba with newly prepared 1:1000 dilution | + | (from gelex)<br /> |
- | <br>*test digest of plasmidpreparations from today | + | -pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR |
- | ->see picture<br> | + | purification)</small><br /> |
- | | + | <br /> |
- | <h3>09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika </h3> | + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | <br>*Phage ss DNA<br> | + | <li>transformation of ligations |
- | -Over night in 4°C room precipitated phages: centrifuged for 20 min at 5000rpm and 4°C<br> | + | </li> |
- | -Discard supernatant, resuspended pellet in 2ml TBS (no pellet recognized)<br> | + | <li>inoculation of pJS419_HisDigSplitFoka in LB + |
- | -Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at 13000rpm<br> | + | chloramphenicol |
- | -Decant the supernatant into new eppis, precipitate with 170µl PEG/NaCl and leave for 1 hour on ice<br> | + | </li> |
- | | + | <li>new two step PCR with all three plasmids for Fos construct |
- | <br>*Miniprep | + | (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution |
- | - pJS 419-his-dig-split-foka (clon 1+2)<br> | + | and preparative agarose gel |
- | - Glycerolstock of clon1 and 2<br> | + | ->interpretation: no band of 921bp was visible</li> |
- | - 300µl Glycerol<br> | + | </ul> |
- | - 700 µl culture<br> | + | <div style="margin-left: 40px;"><br /> |
- | | + | </div> |
- | - Pellets from clones 3,5,6<br> | + | <ul style="margin-left: 40px;"> |
- | | + | <li>one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and |
- | Nanodrop data:<br> | + | preparative agarose gel |
- | pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl<br> | + | ->see picture</li> |
- | pJS 419-his-dig-split-foka-clon2 =468 ng/µl<br> | + | </ul> |
- | | + | <div style="margin-left: 40px;"><small>->interpretation: |
- | <br>*Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room | + | pMAFos and pMASplitFoka showed bands of the |
- | <br>*send to sequencing:<br> | + | right |
- | pMA Dig Plasmidprep 22.07.09 Timo1<br> | + | size (230bp and 657bp respectively), of pMADsbA no product was visible</small><br /> |
- | pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br> | + | <br /> |
- | pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br> | + | </div> |
- | pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4<br> | + | <ul style="margin-left: 40px;"> |
- | pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5<br> | + | <li>new one step PCR of pMADsba with newly prepared 1:1000 |
- | pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6<br> | + | dilution |
- | pMA Cat plasmidprep clone1 08.10.09 Timo7<br> | + | </li> |
- | pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8<br> | + | <li>test digest of plasmidpreparations from today |
- | pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9<br> | + | ->see picture</li> |
- | pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12<br> | + | </ul> |
- | | + | <h3 style="margin-left: 40px;">09.10.09 Manu, Julia, Caro, |
- | <br>* concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation | + | Laura, Christopherus, Hannes, |
- | <br>* electrical trafo of the 449 ago phagmid bibliothek into XL1 | + | Timo, Max, Anika </h3> |
- | <br>* prepaired immutubes with streptavidin for ago phages test panning tomorrow | + | <div style="margin-left: 40px;"><br /> |
- | | + | </div> |
- | | + | <ul style="margin-left: 40px;"> |
- | | + | <li>Phage ss DNA</li> |
- | <h3>10.10.09 Julia, Caro, Laura, Christoph</h3> | + | </ul> |
- | | + | <div style="margin-left: 40px;"><small>-Over night |
- | <br>*Measured OD600 of over night ER2738 culture abs.1)0.218 (1:10);2)0.23 (1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet<br> | + | in 4°C room precipitated phages: centrifuged for 20 |
- | <br>*Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM | + | min at 5000rpm and 4°C<br /> |
- | <br>*Growed Er2738 up to OD600 abs.0.6 8x 250ml in 1L Erlenmeyer flasks and infected with 15µl M13 phage, shake for 4 hours at 37°C | + | -Discard supernatant, resuspended pellet in 2ml TBS (no pellet |
- | <br>*Decanted to 16x50ml falcon tubes, centrifuged for 20min at 5000rpm and 4°C, decanted supernatant into new 16x50ml falcons with each 7ml PEG/NaCl | + | recognized)<br /> |
- | <br>*Precipitaion over night in 4°C room | + | -Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at |
- | <br>*plasmidpreparation of | + | 13000rpm<br /> |
- | - pBADQuick clones 1-6<br> | + | -Decant the supernatant into new eppis, precipitate with |
- | - pBADT4 clones 1-6<br> | + | 170µl PEG/NaCl and leave for 1 hour on ice</small><br /> |
- | - pMAYFP clone 1-2<br> | + | <br /> |
- | - pMACFP clone 1<br> | + | </div> |
- | - pMASplitlinker clones 1-2<br> | + | <ul style="margin-left: 40px;"> |
- | - HisTag clones 1-2 (but test digest was negative -> thrown away)<br< | + | <li>Miniprep |
- | <br>*digest for recloning of pMA constructs: | + | - pJS 419-his-dig-split-foka (clon 1+2)</li> |
- | | + | </ul> |
- | pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from 10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09, pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10µl<br> | + | <div style="margin-left: 40px;"><small>- |
- | Enzymes: 1µl of NgoMIVI and 1.5µl PstI<br> | + | Glycerolstock of clon1 and 2<br /> |
- | Buffer: 3µl buffer 1<br> | + | - 300µl Glycerol<br /> |
- | BSA (10fold): 3µl<br> | + | - 700 µl culture<br /> |
- | Water: 10,5µl<br> | + | - Pellets from clones 3,5,6<br /> |
- | | + | Nanodrop data:<br /> |
- | pMAHisDig clone 2, prep from 24.08.09, pMAStrepDig clone 2, prep from 25.09., pExStrepDig clone 2, prep from 25.09.09, pMABB057 from 01.10.09: 10µl each<br> | + | pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl<br /> |
- | Enzymes: 1µl of AgeI and 1.5µl PstI<br> | + | pJS 419-his-dig-split-foka-clon2 =468 ng/µl<br /> |
- | Buffer: 3µl buffer 1<br> | + | </small><br /> |
- | BSA (10fold): 3µl<br> | + | </div> |
- | Water: 10,5µl<br> | + | <ul style="margin-left: 40px;"> |
- | <br>*PCR assembly with pMADsbA, different approaches (Taq Polymerase or Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and pMAFos together | + | <li>Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml |
- | <br>*analytical gel of PCRs -> didn't work | + | Tet, over |
- | <br>*preparative gels of digests | + | night in 37°C room |
- | -> see picture | + | </li> |
- | -> no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more | + | <li>send to sequencing:</li> |
- | <br>*ligation of | + | </ul> |
- | -pMAStrepDigShortLiFoka<br> | + | <div style="margin-left: 40px;"><small>pMA Dig |
- | -pMAStrepDigMiddleLiFoka<br> | + | Plasmidprep 22.07.09 Timo1<br /> |
- | -pMAStrepDigLongLiFoka<br> | + | pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br /> |
- | -pExStrepDigShortLiFoka<br> | + | pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br /> |
- | -pExStrepDigMiddleLiFoka<br> | + | pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4<br /> |
- | -pExStrepDigLongLiFoka<br> | + | pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5<br /> |
- | -pMAHisDigShortLiFoka<br> | + | pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6<br /> |
- | -pMAHisDigMiddleLiFoka<br> | + | pMA Cat plasmidprep clone1 08.10.09 Timo7<br /> |
- | -pMAHisDigLongLiFoka<br> | + | pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8<br /> |
- | -pMACATNd4 (MQI)<br> | + | pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9<br /> |
- | -pMACATNd4 (MQII)<br> | + | pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12</small><br /> |
- | -pMA Kontrolle (M)<br> | + | <br /> |
- | -> in XLBlue<br> | + | </div> |
- | - pEx_CATNd4 (EQI)<br> | + | <ul style="margin-left: 40px;"> |
- | - pEx_CATNd4 (EQII)<br> | + | <li> concentrated pET39b+ ssDNA via sodiumacetat and |
- | - pEx_Kontrolle(E)<br> | + | ethanol precipitation |
- | ->RV308 | + | </li> |
- | Insert: 6µl <br> | + | <li> electrical trafo of the 449 ago phagmid |
- | vector: 3µl <br> | + | bibliothek into XL1 |
- | Ouick ligase buffer: 10µl <br> | + | </li> |
- | Quick ligase: 1µl <br> | + | <li> prepaired immutubes with streptavidin for ago |
- |
| + | phages test panning |
- | <br>*transformation of ligations in XBL on LB/Amp/1%glucose plates | + | tomorrow |
- | <br>*cotransformation of | + | </li> |
- | <br>*testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture<br> | + | </ul> |
- | [[Image:Freiburg09 101009pet39b+ssdnaagoassay.JPG|none|thumb|gele of the AGO pETb+ ssDNA cleavage assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA|400x400px]]
| + | <h3 style="margin-left: 40px;">10.10.09 Julia, Caro, |
- | <br>*cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates | + | Laura, Christoph</h3> |
- | <br>*inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09 | + | <div style="margin-left: 40px;"><br /> |
- | | + | </div> |
- | <h3> 11.10.09, Timo, Hannes, Max, Anika</h3> | + | <ul style="margin-left: 40px;"> |
- | | + | <li>Measured OD600 of over night ER2738 culture abs.1)0.218 |
- | <br>* Digestion of | + | (1:10);2)0.23 |
- | 1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI<br> | + | (1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet</li> |
- | 2.pMAFos_HlsbZip, XbaI - AgI<br> | + | <li>Measured OD600 of Christophs culture:abs. 0.37 (1:10), |
- | 3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br> | + | diluted to |
- | 4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br> | + | abs.0.07 in 60ml DYT+Tet+CM |
- | 5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br> | + | </li> |
- | 6.pMA_BB057, XbaI - PstI (01.10.09)<br> | + | <li>Growed Er2738 up to OD600 abs.0.6 8x 250ml in 1L Erlenmeyer |
- | <br>*gel extraction of | + | flasks and |
- | - pMA<br> | + | infected with 15µl M13 phage, shake for 4 hours at |
- | - pEx<br> | + | 37°C |
| + | </li> |
| + | <li>Decanted to 16x50ml falcon tubes, centrifuged for 20min at |
| + | 5000rpm |
| + | and 4°C, decanted supernatant into new 16x50ml falcons with |
| + | each 7ml |
| + | PEG/NaCl |
| + | </li> |
| + | <li>Precipitaion over night in 4°C room |
| + | </li> |
| + | <li>plasmidpreparation of |
| + | - pBADQuick clones 1-6</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small> |
| + | - pBADT4 clones 1-6<br /> |
| + | - pMAYFP clone 1-2<br /> |
| + | - pMACFP clone 1<br /> |
| + | - pMASplitlinker clones 1-2<br /> |
| + | - HisTag clones 1-2 (but test digest was negative -> thrown away)<br /> |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>digest for recloning of pMA constructs: |
| + | pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka |
| + | clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from |
| + | 10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09, |
| + | pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>Enzymes: |
| + | 1µl of NgoMIVI and 1.5µl PstI<br /> |
| + | Buffer: 3µl buffer 1<br /> |
| + | BSA (10fold): 3µl<br /> |
| + | Water: 10,5µl<br /> |
| + | pMAHisDig clone 2, prep from 24.08.09, pMAStrepDig clone 2, prep from |
| + | 25.09., pExStrepDig clone 2, prep from 25.09.09, pMABB057 from |
| + | 01.10.09: 10µl each<br /> |
| + | Enzymes: 1µl of AgeI and 1.5µl PstI<br /> |
| + | Buffer: 3µl buffer 1<br /> |
| + | BSA (10fold): 3µl<br /> |
| + | Water: 10,5µl</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>PCR assembly with pMADsbA, different approaches (Taq |
| + | Polymerase or |
| + | Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and |
| + | pMAFos together </li> |
| + | <li>analytical gel of PCRs -> didn't work </li> |
| + | <li>preparative gels of digests |
| + | -> see picture |
| + | -> no inserts with pExDSba_Foka constructs because they have no |
| + | NgoMIV site any more |
| + | </li> |
| + | <li>ligation of -pMAStrepDigShortLiFoka</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-pMAStrepDigMiddleLiFoka<br /> |
| + | -pMAStrepDigLongLiFoka<br /> |
| + | -pExStrepDigShortLiFoka<br /> |
| + | -pExStrepDigMiddleLiFoka<br /> |
| + | -pExStrepDigLongLiFoka<br /> |
| + | -pMAHisDigShortLiFoka<br /> |
| + | -pMAHisDigMiddleLiFoka<br /> |
| + | -pMAHisDigLongLiFoka<br /> |
| + | -pMACATNd4 (MQI)<br /> |
| + | -pMACATNd4 (MQII)<br /> |
| + | -pMA Kontrolle (M)<br /> |
| + | -> in XLBlue<br /> |
| + | - pEx_CATNd4 (EQI)<br /> |
| + | - pEx_CATNd4 (EQII)<br /> |
| + | - pEx_Kontrolle(E)<br /> |
| + | ->RV308 |
| + | Insert: 6µl <br /> |
| + | vector: 3µl <br /> |
| + | Ouick ligase buffer: 10µl <br /> |
| + | Quick ligase: 1µl </small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>transformation of ligations in XBL on LB/Amp/1%glucose |
| + | plates |
| + | </li> |
| + | <li>cotransformation of |
| + | </li> |
| + | <li>testdigest of the pET39b+ ssDNA. looks like a success... |
| + | see 4pk in |
| + | picture</li> |
| + | </ul> |
| + | <table |
| + | style="text-align: left; width: 389px; height: 342px; margin-left: 40px;" |
| + | border="0" cellpadding="0" cellspacing="0"> |
| + | <tbody> |
| + | <tr> |
| + | <td><img class="art-article" |
| + | style="width: 350px; height: 280px;" alt="" |
| + | src="https://static.igem.org/mediawiki/2009/d/d3/Freiburg09_101009pet39b%2Bssdnaagoassay.JPG" /></td> |
| + | </tr> |
| + | <tr> |
| + | <td><small>gele of the AGO pETb+ ssDNA cleavage |
| + | assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <div style="margin-left: 40px;"><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>cotransformation of pExHisFluASplitFoki and |
| + | pJSStrepDigSplitFoka in |
| + | XBL on LB/Amp/CM/1%glucose plates |
| + | </li> |
| + | <li>inoculation of pExDsbAStrepDigSplitFoka and |
| + | pExDsbAHisDigSplitFoka, |
| + | glystock from 02.10.09 |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;"> 11.10.09, Timo, Hannes, |
| + | Max, Anika</h3> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Digestion of </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>1.pEXHisFluA |
| + | (27.09.09, Klon1), XbaI - PstI<br /> |
| + | 2.pMAFos_HlsbZip, XbaI - AgI<br /> |
| + | 3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br /> |
| + | 4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br /> |
| + | 5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br /> |
| + | 6.pMA_BB057, XbaI - PstI (01.10.09)</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>gel extraction of |
| + | - pMA</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- pEx<br /> |
| - ... | | - ... |
- | | + | </small><br /> |
- | <br>* Testdigestion of | + | </div> |
- | 7.pMA_YFP 2 (10.10.09, Caro)<br> | + | <ul style="margin-left: 40px;"> |
- | 8.pMA_CFP (10.10.09, Caro)<br> | + | <li> Testdigestion of </li> |
- | 9.pMASplitLi1 (10.10.09, Caro)<br> | + | </ul> |
- | 10.pMASplitLi2 (10.10.09, Caro)<br> | + | <div style="margin-left: 40px;"><small>7.pMA_YFP 2 |
- | ...image.. | + | (10.10.09, Caro)<br /> |
- | <br>* 1% Agarosegel of constructs above | + | 8.pMA_CFP (10.10.09, Caro)<br /> |
- | <br>* Phage breeding day 2 | + | 9.pMASplitLi1 (10.10.09, Caro)<br /> |
- | <br>* Starter culture of pEx_DsbA_StrepDigSplitFoka (Bl21de3) | + | 10.pMASplitLi2 (10.10.09, Caro)<br /> |
- | | + | ...image.. </small><br /> |
- | <br>* plasmid prep of pEx_DsbA_StrepDigSplitFoka | + | </div> |
- | <br>* plasmid prep of pEx_HisDigSplitFoka | + | <ul style="margin-left: 40px;"> |
- | | + | <li> 1% Agarosegel of constructs above |
- | <br>* M13 ssDNA produced with bacterial of Julia and tried another variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected with <br> M13phage particles and let it grow for 2h. After this followed the qiagen M13 protocol for M13Dna | + | </li> |
- | | + | <li> Phage breeding day 2 |
- | <h3> 12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia, Timo, Gerrit</h3> | + | </li> |
- | <br>* protein expression of pEx_DsbA_StrepDigSplitFoka (periplasm) in BL21de3 at 22°C. | + | <li> Starter culture of pEx_DsbA_StrepDigSplitFoka |
- | <br>* made 5 litres of DYT | + | (Bl21de3) |
- | <br>*digest of pMASplitFoka clone 1 and 2 from 04.10.09 | + | </li> |
- | DNA:10µl | + | <li> plasmid prep of pEx_DsbA_StrepDigSplitFoka |
- | Enzymes: 1µl of NgoMIVI and 1.5µl PstI<br> | + | </li> |
- | Buffer: 3µl buffer 1<br> | + | <li> plasmid prep of pEx_HisDigSplitFoka |
- | BSA (100fold): 0.5µl<br> | + | </li> |
- | Water: 14.5µl<br> | + | <li> M13 ssDNA produced with bacterial of Julia and |
- | <br>* Plasmidprep. of: <br> | + | tried another |
- | 1.pMAFokA (old)<br> | + | variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected |
- | 2.pMA-CAT Ndelta4 (MQII)1<br> | + | withM13phage particles and let it grow for 2h. After this followed the |
- | 3.pMA-CAT Ndelta4 (MQII)2<br> | + | qiagen M13 protocol for M13Dna |
- | 4.pMA-CAT Ndelta4 (MQII)3<br> | + | </li> |
- | 5.pMA-CAT Ndelta4 (MQII)4<br> | + | </ul> |
- | 6.pMA-CAT Ndelta4 (MQII)5<br> | + | <h3 style="margin-left: 40px;"> 12.10.09, Laura, Caro, |
- | 7.pMA-CAT Ndelta4 (MQII)6<br> | + | Christoph, Anika, Hannes, Julia, |
- | 8.pMAFoka clone 1 from 04.10.<br> | + | Timo, Gerrit</h3> |
- | <br>*Made new BB-AGO PCR using digested AGO Gene without the vector-> still no expected bands to be seen... | + | <div style="margin-left: 40px;"><br /> |
- | <br>*Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay | + | </div> |
- | <br>*Made digest of errorprone PCR product of the AGO-G3P constructs (from 02.10.; one with, one without Amber) via NheI and SfiI to gain new Phagmid library | + | <ul style="margin-left: 40px;"> |
- | | + | <li> protein expression of pEx_DsbA_StrepDigSplitFoka |
- | <br>*Send to sequencing:[[Protocols#DNA_Sequencing]]<br> | + | (periplasm) in |
- | Julia 1-6:<br> | + | BL21de3 at 22°C. |
- | 1)pBADQuick clone 3<br> | + | </li> |
- | 2)pBAD T4 clone 2<br> | + | <li> made 5 litres of DYT |
- | 3)pBAD T4 clone 1<br> | + | </li> |
- | 4)pMA YFP clone 1<br> | + | <li>digest of pMASplitFoka clone 1 and 2 from 04.10.09 |
- | 5)pMA CFP clone 1<br> | + | DNA:10µl |
- | 6)pMA Split Linker clone 2<br><br> | + | Enzymes: 1µl of NgoMIVI and 1.5µl PstI</li> |
- | | + | </ul> |
| + | <div style="margin-left: 40px;"><small>Buffer: |
| + | 3µl buffer 1<br /> |
| + | BSA (100fold): 0.5µl<br /> |
| + | Water: 14.5µl<br /> |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li> Plasmidprep. of: </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>1.pMAFokA |
| + | (old)<br /> |
| + | 2.pMA-CAT Ndelta4 (MQII)1<br /> |
| + | 3.pMA-CAT Ndelta4 (MQII)2<br /> |
| + | 4.pMA-CAT Ndelta4 (MQII)3<br /> |
| + | 5.pMA-CAT Ndelta4 (MQII)4<br /> |
| + | 6.pMA-CAT Ndelta4 (MQII)5<br /> |
| + | 7.pMA-CAT Ndelta4 (MQII)6<br /> |
| + | 8.pMAFoka clone 1 from 04.10.</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Made new BB-AGO PCR using digested AGO Gene without the |
| + | vector-> still no expected bands to be seen... |
| + | </li> |
| + | <li>Made new PCR to generate more pET39b+ ssDNA because |
| + | yesterdays had |
| + | insufficient concentration for the AGO cleavage assay </li> |
| + | <li>Made digest of errorprone PCR product of the AGO-G3P |
| + | constructs |
| + | (from 02.10.; one with, one without Amber) via NheI and SfiI to gain |
| + | new Phagmid library |
| + | </li> |
| + | <li>Send to sequencing:[[Protocols#DNA_Sequencing]]</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>Julia 1-6:<br /> |
| + | 1)pBADQuick clone 3<br /> |
| + | 2)pBAD T4 clone 2<br /> |
| + | 3)pBAD T4 clone 1<br /> |
| + | 4)pMA YFP clone 1<br /> |
| + | 5)pMA CFP clone 1<br /> |
| + | 6)pMA Split Linker clone 2<br /> |
| + | <br /> |
| Gerrit1: pMA_cat-Nd4 | | Gerrit1: pMA_cat-Nd4 |
- | | + | </small><br /> |
- | <br>*Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for Panning Simulation for tomorrow | + | </div> |
- | <br>*made chemical competent cells of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki | + | <ul style="margin-left: 40px;"> |
- | ->will prepare electrocompetent cells tomorrow<br> | + | <li>Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for |
- | <br>*inoculation of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp | + | Panning |
- | <br>*PCR of pMADsba repeated with different (higher) template dilutions | + | Simulation for tomorrow |
- | ->analytical gel (see picture)<br> | + | </li> |
- | ->didn't work again<br> | + | <li>made chemical competent cells of cotransformed XBlue with |
- | <br>*poured IPTG/XGal plates for in vivo plaque assay test run | + | pJS419HisDigSplitFoka, pExHisFluASplitFoki |
- | | + | ->will prepare electrocompetent cells tomorrow</li> |
- | <br>*transformation of:<br> | + | <li>inoculation of cotransformed XBlue with |
- | - pBAD_FokA (Quick)<br> | + | pJS419HisDigSplitFoka, |
- | - pBAD_CAT(Quick)<br> | + | pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp |
- | - pBAD_FokA (T4)<br> | + | </li> |
- | - pBAD_CAT (T4)<br> | + | <li>PCR of pMADsba repeated with different (higher) template |
- | - pEX_FokA+YFP<br> | + | dilutions |
- | - pEX_DsbA+FokA+YFP<br> | + | ->analytical gel (see picture)</li> |
- | into BL21 de3 gold<br> | + | </ul> |
- | Plates: LB+ AMP +1%Glucose<br> | + | <div style="margin-left: 40px;"><small> |
- | <br>*cultivation of | + | ->didn't work again</small><br /> |
- | pma strep dig split foka<br> | + | <br /> |
- | pma long linker<br> | + | </div> |
- | pma his flua split foki<br> | + | <ul style="margin-left: 40px;"> |
- | | + | <li>poured IPTG/XGal plates for in vivo plaque assay test run |
- | | + | </li> |
- | <h3> 13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia</h3> | + | <li>transformation of:</li> |
- | | + | </ul> |
- | <br>*Miniprep | + | <div style="margin-left: 40px;"><small>- pBAD_FokA |
- | | + | (Quick)<br /> |
- | -pMA-fos 1<br> | + | - pBAD_CAT(Quick)<br /> |
- | -pMA-fos 2<br> | + | - pBAD_FokA (T4)<br /> |
- | -pMA his flua split fokI<br> | + | - pBAD_CAT (T4)<br /> |
- | - pMA strep dig split foka <br> | + | - pEX_FokA+YFP<br /> |
- | -pMA long linker<br> | + | - pEX_DsbA+FokA+YFP<br /> |
- | | + | into BL21 de3 gold<br /> |
- | <br>*testdigest of Plasmidprep from today | + | Plates: LB+ AMP +1%Glucose<br /> |
- | | + | </small><br /> |
- | 15 µl for 6µl loading dye | + | </div> |
- | -buffer 2 1µl<br> | + | <ul style="margin-left: 40px;"> |
- | -xbaI 0.5µl<br> | + | <li>cultivation of |
- | -pstI 0.75µl<br> | + | pma strep dig split foka</li> |
- | -DNA 2 µl<br> | + | </ul> |
- | -water 9.75µl<br> | + | <div style="margin-left: 40px;"><small>pma long |
- | -BSA 1µl<br> | + | linker<br /> |
- | | + | pma his flua split foki</small><br /> |
- | --> Gelbild | + | </div> |
- | | + | <h3 style="margin-left: 40px;"> 13.10.09, Laura, Caro, |
| + | Christoph,Manu, Hannes, Julia</h3> |
| + | <div style="margin-left: 40px;"><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Miniprep |
| + | -pMA-fos 1</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-pMA-fos 2<br /> |
| + | -pMA his flua split fokI<br /> |
| + | - pMA strep dig split foka <br /> |
| + | -pMA long linker</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>testdigest of Plasmidprep from today |
| + | 15 µl for 6µl loading dye |
| + | -buffer 2 1µl</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-xbaI |
| + | 0.5µl<br /> |
| + | -pstI 0.75µl<br /> |
| + | -DNA 2 µl<br /> |
| + | -water 9.75µl<br /> |
| + | -BSA 1µl<br /> |
| + | --> Gelbild |
| - Glycerolstocks are in in box of 5/10/2009 | | - Glycerolstocks are in in box of 5/10/2009 |
- | <br>*made electrocompetent XL1 Blue cotransformed cells (pExHisFluASplitFoki and pJS419HisDigSplitFoka) | + | </small><br /> |
- | ->stored in -80°C | + | </div> |
- | <br>*culture of ER2738 in LB Tet for cotransformation in afternoon | + | <ul style="margin-left: 40px;"> |
- | <br>*test in vivo assay | + | <li>made electrocompetent XL1 Blue cotransformed cells |
- | - electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV<br> | + | (pExHisFluASplitFoki and pJS419HisDigSplitFoka) |
- | - 1.5h on 37°C shaker at 750rpm<br> | + | ->stored in -80°C |
- | - precipitation of phages<br> | + | </li> |
- | - infection of ER2738 with different dilutions of phages<br> | + | <li>culture of ER2738 in LB Tet for cotransformation in |
- | - mixed cells with Top agar and plated them on IPTG/XGAL plates -> 37° shaker<br> | + | afternoon |
- | <br>*inoculation of | + | </li> |
- | -pMASplitFoka clone 1 from prep 04.10.09<br> | + | <li>test in vivo assay - electroporation with new |
- | -pMAStrepDig clone 2 from prep 25.09.09<br> | + | electrocompetent cells |
- | -pMAMiddleFoka clone 2 from prep 04.10.09<br> | + | and M13 ssDNA at 1.7kV</li> |
- | -pMAShortFoka clone 2 from prep 04.10.09<br> | + | </ul> |
- | -pMAFoka clone 1 from prep 04.10.09<br> | + | <div style="margin-left: 40px;"><small>- 1.5h on |
- | -pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09<br> | + | 37°C shaker at 750rpm<br /> |
- | -pMALongFoka clone1 prep from 10.09.09<br> | + | - precipitation of phages<br /> |
- | -pMAFos 13.10.09<br> | + | - infection of ER2738 with different dilutions of phages<br /> |
- | -pBAD_CAT 13.10.09<br> | + | - mixed cells with Top agar and plated them on IPTG/XGAL plates |
- | -pBAD_Foka 13.10.09<br> | + | -> 37° shaker</small><br /> |
- | -pMADsbAHisDigSplitFoka 13.10.09<br> | + | <br /> |
- | -pMADsbAStrepDigSplitFoka 13.10.09<br> | + | </div> |
- | -pExFosSplitFoka 13.10.09<br> | + | <ul style="margin-left: 40px;"> |
- | -pMAFosSplitFoka 13.10.09<br> | + | <li>inoculation of |
- | <br>*protein purification of DsbA_StrepDigSplitFoka with Strep column | + | -pMASplitFoka clone 1 from prep 04.10.09</li> |
- | | + | </ul> |
- | <br>*digestion, 1% agarose gel and gel extraction of | + | <div style="margin-left: 40px;"><small>-pMAStrepDig |
- | - pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI<br> | + | clone 2 from prep 25.09.09<br /> |
- | - pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI<br> | + | -pMAMiddleFoka clone 2 from prep 04.10.09<br /> |
- | - pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI => wasn't ok on the gel, inoculated clone 1<br> | + | -pMAShortFoka clone 2 from prep 04.10.09<br /> |
- | - pMA-ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br> | + | -pMAFoka clone 1 from prep 04.10.09<br /> |
- | - pMA-MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br> | + | -pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09<br /> |
- | - pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br> | + | -pMALongFoka clone1 prep from 10.09.09<br /> |
- | - pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br> | + | -pMAFos 13.10.09<br /> |
- | - pEx-Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI<br> | + | -pBAD_CAT 13.10.09<br /> |
- | - pEx-Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI<br> | + | -pBAD_Foka 13.10.09<br /> |
| + | -pMADsbAHisDigSplitFoka 13.10.09<br /> |
| + | -pMADsbAStrepDigSplitFoka 13.10.09<br /> |
| + | -pExFosSplitFoka 13.10.09<br /> |
| + | -pMAFosSplitFoka 13.10.09</small><br /> |
| + | <br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>protein purification of DsbA_StrepDigSplitFoka with Strep |
| + | column |
| + | </li> |
| + | <li>digestion, 1% agarose gel and gel extraction of |
| + | - pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI<br /> |
| + | - pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI => |
| + | wasn't ok on the gel, inoculated clone 1<br /> |
| + | - pMA-ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br /> |
| + | - pMA-MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br /> |
| + | - pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br /> |
| + | - pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br /> |
| + | - pEx-Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI<br /> |
| + | - pEx-Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI<br /> |
| - pEx-Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI | | - pEx-Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI |
- | - pMA (01.10., c=500ng/µl) with XbaI + AgeI | + | - pMA (01.10., c=500ng/µl) with XbaI + AgeI |
- | - pMA (01.10., c=500ng/µl) with EcoRI + SpeI | + | - pMA (01.10., c=500ng/µl) with EcoRI + SpeI |
- | | + | </small><br /> |
- | | + | </div> |
- | <br>*ligations: | + | <ul style="margin-left: 40px;"> |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI<br> | + | <li>ligations: |
- | Insert: 6 µl LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI<br> | + | Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | SpeI</li> |
- | QuickLigase: 1 µl<br><br> | + | </ul> |
- | | + | <div style="margin-left: 40px;"><small>Insert: 6 |
- | Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br> | + | µl LongLinker-FokA (10.09., clone 1) with EcoRI + |
- | Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br> | + | SpeI<br /> |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | QuickLigase: 1 µl<br><br> | + | QuickLigase: 1 µl<br /> |
- | | + | <br /> |
- | Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br> | + | Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br /> |
- | Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br> | + | Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | PstI<br /> |
- | QuickLigase: 1 µl<br><br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | | + | QuickLigase: 1 µl<br /> |
- | Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br> | + | <br /> |
- | Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br> | + | Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br /> |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV |
- | QuickLigase: 1 µl<br><br> | + | + PstI<br /> |
- | | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br> | + | QuickLigase: 1 µl<br /> |
- | Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br> | + | <br /> |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br /> |
- | QuickLigase: 1 µl<br><br> | + | Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + |
- | | + | PstI<br /> |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | Insert: 6 µl Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI<br> | + | QuickLigase: 1 µl<br /> |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | <br /> |
- | QuickLigase: 1 µl<br><br> | + | Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br /> |
- | | + | Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br> | + | + PstI<br /> |
- | Insert: 6 µl Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI<br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | QuickLigase: 1 µl<br /> |
- | QuickLigase: 1 µl<br><br> | + | <br /> |
- | | + | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br> | + | AgeI<br /> |
- | Insert: 6 µl Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI<br> | + | Insert: 6 µl Strep-FluA-MiddleLinker-FokI (08.10., clone 1) |
- | Buffer: 10 µl, QuickLigase Buffer NEB<br> | + | with XbaI + AgeI<br /> |
- | QuickLigase: 1 µl<br><br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | | + | QuickLigase: 1 µl<br /> |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br> | + | <br /> |
- | Insert: 2 µl ShortLinker, hybridised, with XbaI + AgeI<br> | + | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + |
- | Buffer: 14 µl, QuickLigase Buffer NEB<br> | + | AgeI<br /> |
- | QuickLigase: 1 µl<br><br> | + | Insert: 6 µl Strep-FluA-MiddleLinker-FokA (08.10., clone 1) |
- | | + | with XbaI + AgeI<br /> |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | Insert: 2 µl MiddleLinker, hybridised, with XbaI + AgeI<br> | + | QuickLigase: 1 µl<br /> |
- | Buffer: 14 µl, QuickLigase Buffer NEB<br> | + | <br /> |
- | QuickLigase: 1 µl<br><br> | + | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + |
- | | + | AgeI<br /> |
- | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br> | + | Insert: 6 µl Strep-FluA-LongLinker-FokI (08.10., Klon 1) with |
- | Insert: 2 µl LongLinker, hybridised, with XbaI + AgeI<br> | + | XbaI + AgeI<br /> |
- | Buffer: 14 µl, QuickLigase Buffer NEB<br> | + | Buffer: 10 µl, QuickLigase Buffer NEB<br /> |
- | QuickLigase: 1 µl<br><br> | + | QuickLigase: 1 µl<br /> |
- | | + | <br /> |
- | <br>*did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc | + | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + |
- | | + | AgeI<br /> |
- | | + | Insert: 2 µl ShortLinker, hybridised, with XbaI + AgeI<br /> |
- | <br>*ELISA | + | Buffer: 14 µl, QuickLigase Buffer NEB<br /> |
- | -Blocking<br> | + | QuickLigase: 1 µl<br /> |
- | -Washing with TBST-EDTA 5x<br> | + | <br /> |
- | -Loaded immunotubes (1.5ml) and wells 100µl each with 0.5mM biotin-target DNA<br>
| + | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + |
- | -Incubation at 20°C in shaking<br>
| + | AgeI<br /> |
- | -Incubatet Phages with Oligo for 30min at 55°c waterbath<br>
| + | Insert: 2 µl MiddleLinker, hybridised, with XbaI + AgeI<br /> |
- | -Washing of wells and immunotubes with TBST-EDTA 5x<br>
| + | Buffer: 14 µl, QuickLigase Buffer NEB<br /> |
- | -Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1 phage+Oligo and 1+control as a control<br>
| + | QuickLigase: 1 µl<br /> |
- | -Incubation in shaker at 20°C<br>
| + | <br /> |
- | -Loaded immunotubes (each 1.5 ml) with 50µl P1, 25µl 449Phage+ Oligo in TBST-EDTA<br>
| + | Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + |
- | -Incubation in shaker at 20°C<br>
| + | AgeI<br /> |
- | -Washing of wells and immunotubes with TBST-EDTA 5x
| + | Insert: 2 µl LongLinker, hybridised, with XbaI + AgeI<br /> |
- | -Loaded 100µl/well anti-M13 ab(1:1000), incubated for 1 hour at 20°C in shaker<br> | + | Buffer: 14 µl, QuickLigase Buffer NEB<br /> |
- | -Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS 3x<br>
| + | QuickLigase: 1 µl<br /> |
- | -2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS-T 3x<br>
| + | </small><br /> |
- | -3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis<br>
| + | <br /> |
- | | + | </div> |
- | <br>*Made new "AGO BB PCR" this time just using the ad-on-tail-primer for the bb pre- and suffix, so that hte EcoRI site should remain. Still the same not fitting bands were obtained-> the problem might be caused by multimerising of the primers, even so we were not able to predict any of tose using vectir nti or compairable programms... | + | <ul style="margin-left: 40px;"> |
- | <br>* AGO assay using different targets-> were not able to reproduce the presumed cutting event from Saturdays assay probably due to the lower DNA concentration | + | <li>did transformations of the ligations in RV308, plated on |
- | <br>* Made ELISA with strep-biotinylated target oligo coated surface-> no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS | + | LB-Amp + 1% |
- | <br>* Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I | + | Gluc |
- | <br>* Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence | + | </li> |
- | | + | <li>ELISA -Blocking</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>Washing with |
| + | TBST-EDTA 5x<br /> |
| + | Loaded immunotubes (1.5ml) and wells 100µl each with 0.5mM |
| + | biotin-target DNA<br /> |
| + | Incubation at 20°C in shaking<br /> |
| + | Incubatet Phages with Oligo for 30min at 55°c waterbath<br /> |
| + | Washing of wells and immunotubes with TBST-EDTA 5x<br /> |
| + | Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1 |
| + | phage+Oligo and 1+control as a control<br /> |
| + | Incubation in shaker at 20°C<br /> |
| + | Loaded immunotubes (each 1.5 ml) with 50µl P1, |
| + | 25µl 449Phage+ Oligo in TBST-EDTA<br /> |
| + | Incubation in shaker at 20°C<br /> |
| + | Washing of wells and immunotubes with TBST-EDTA 5x |
| + | -Loaded 100µl/well anti-M13 ab(1:1000), incubated for 1 hour |
| + | at 20°C in shaker<br /> |
| + | Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20 |
| + | min, decanted supernatant into eppis, washed with TBS 3x<br /> |
| + | 2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted |
| + | supernatant into eppis, washed with TBS-T 3x<br /> |
| + | 3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis<br /> |
| + | </small><br /> |
| + | </div> |
| + | <ul style="margin-left: 40px;"> |
| + | <li>Made new "AGO BB PCR" this time just using the |
| + | ad-on-tail-primer |
| + | for the bb pre- and suffix, so that hte EcoRI site should remain. Still |
| + | the same not fitting bands were obtained-> the problem might be |
| + | caused by multimerising of the primers, even so we were not able to |
| + | predict any of tose using vectir nti or compairable programms... |
| + | </li> |
| + | <li>AGO assay using different targets-> were not able to |
| + | reproduce the presumed cutting event from Saturdays assay probably due |
| + | to the lower DNA concentration </li> |
| + | <li>Made ELISA with strep-biotinylated target oligo coated |
| + | surface-> no binding of AGO bearing phages was detected using |
| + | anti-M13 HRP and ABTS |
| + | </li> |
| + | <li>Made test panning just like the ELISA above and eludet with |
| + | TBS-MnCl2 |
| + | (5mM), then TBST-MnCl2 (5mM) and then with DNase I |
| + | </li> |
| + | <li>Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence</li> |
| + | </ul> |
| | | |
| + | <h3 style="margin-left: 40px;"> 14.10.09, Laura, Caro, |
| + | Christoph, Hannes, Julia, Anika</h3> |
| + | <ul> |
| + | <li> Made LB-medium, 0,7% LB-Agar, 50% Glucose |
| + | * Test expression (15ml) of pBAD_CAT and pBAD_FokA in BL21de3 |
| + | </li> |
| + | </ul> |
| + | <table |
| + | style="border-collapse: collapse; table-layout: fixed; width: 659pt;" |
| + | border="1" cellpadding="0" cellspacing="0" |
| + | width="879"> |
| + | <tbody> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="height: 15pt; width: 282pt;" height="20" |
| + | width="376"><small>OD measurements</small></td> |
| + | <td class="xl6310961" |
| + | style="border-left: medium none; width: 89pt;" width="119"><small>1 |
| + | hour before</small></td> |
| + | <td class="xl6310961" |
| + | style="border-left: medium none; width: 73pt;" width="97"><small>induction</small></td> |
| + | <td class="xl6310961" |
| + | style="border-left: medium none; width: 80pt;" width="107"><small>45min</small></td> |
| + | <td class="xl6310961" |
| + | style="border-left: medium none; width: 68pt;" width="91"><small>1.5h</small></td> |
| + | <td class="xl6310961" |
| + | style="border-left: medium none; width: 67pt;" width="89"><small>2h |
| + | 45min</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small> </small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>induction</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small> </small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small> </small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small> </small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small> </small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>1. |
| + | clone1 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.1</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.74</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.19</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.25</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.34</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>2. |
| + | clone1 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.1</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.67</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.08</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.94</small></td> |
| + | <td class="xl6410961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.20</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>3. |
| + | clone 1 pBAD_CAT control</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.07</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.72</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.08</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.24</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.23</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>4. |
| + | clone2 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.07</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.63</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.96</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.11</small></td> |
| + | <td class="xl6410961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.00</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>5. |
| + | clone2 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.07</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.66</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.06</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.25</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.21</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>6. |
| + | clone2 pBAD_CAT control</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.08</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.72</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.11</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.33</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.25</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>7. |
| + | clone1 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.07</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.67</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.13</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.26</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.07</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>8. |
| + | clone1 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.09</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.67</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.21</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.28</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.22</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>9. |
| + | clone1 pBAD_Foka control</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.09</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.7</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.11</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.38</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.31</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>10. |
| + | clone2 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.07</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.6</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.93</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.16</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.87</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>11. |
| + | clone2 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.07</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.62</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.94</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.18</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.93</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; height: 15pt;" height="20"><small>12. |
| + | clone2 pBAD_Foka control</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.01</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>0.64</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.04</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>1.24</small></td> |
| + | <td class="xl6310961" |
| + | style="border-top: medium none; border-left: medium none;"><small>2.27</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| <br> | | <br> |
| + | <div style="margin-left: 40px;"><small>--> no |
| + | differences between induced and control cells |
| + | <br> |
| + | <br> |
| + | </small> |
| + | <table style="text-align: left; width: 750px;" border="1" |
| + | cellpadding="2" cellspacing="0"> |
| + | <tbody> |
| + | <tr> |
| + | <td><small><img |
| + | style="width: 400px; height: 400px;" alt="" |
| + | src="https://static.igem.org/mediawiki/2009/7/70/Freiburg09_141009_pg_testexpression1015.jpg"></small></td> |
| + | <td><img style="width: 400px; height: 400px;" |
| + | alt="" |
| + | src="https://static.igem.org/mediawiki/2009/1/19/Freiburg09_141009_pg_testexpression2016.jpg"></td> |
| + | </tr> |
| + | <tr> |
| + | <td><small>SDS gel of testexpression with <b>pBAD_CAT</b> |
| + | in BL21de3; lanes: NEB |
| + | prestained protein marker broad range, clone1 pBAD_CAT+0.6mM IPTG+0.2% |
| + | Arabinose, clone1 pBAD_CAT control, clone1 pBAD_CAT+0.6mM IPTG+0.002% |
| + | Arabinose, empty lane, clone2 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, |
| + | clone2 pBAD_CAT control, clone2 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, |
| + | empty lane, NEB prestained protein marker borad range</small></td> |
| + | <td><small>SDS gel of testexpression with <b>pBAD_Foka</b> |
| + | in BL21de3; lanes: NEB |
| + | prestained protein marker broad range, clone1 pBAD_Foka+0.6mM IPTG+0.2% |
| + | Arabinose, clone1 pBAD_Foka control, clone1 pBAD_Foka+0.6mM IPTG+0.002% |
| + | Arabinose, empty lane, clone2 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, |
| + | clone2 pBAD_Foka control, clone2 pBAD_Foka+0.6mM IPTG+0.002% Arabinose</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <small><br> |
| + | </small></div> |
| + | <div style="margin-left: 40px;"><br> |
| </div> | | </div> |
- | <div style="text-align: left;"><br /> | + | <br> |
| + | <ul> |
| + | <li> SDS gel of DsbA_StrepDigSplitFoka purification |
| + | from 13.10.09</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"> |
| + | <table style="text-align: left; width: 750px;" border="1" |
| + | cellpadding="2" cellspacing="0"> |
| + | <tbody> |
| + | <tr> |
| + | <td><img style="width: 400px; height: 400px;" |
| + | alt="" |
| + | src="https://static.igem.org/mediawiki/2009/d/df/Freiburg09_141009_pg_pEx_DsbA_StrepDigSplitFoka_Elution017.jpg"></td> |
| + | <td><img style="width: 400px; height: 400px;" |
| + | alt="" |
| + | src="https://static.igem.org/mediawiki/2009/d/de/Freiburg09_141009_pg_pEx_DsbA_StrepDigSplitFoka2018.jpg"></td> |
| + | </tr> |
| + | <tr> |
| + | <td><small>SDS gel of protein expression and |
| + | purification with Streptag column of <b>pEx_DsbA_StrepDigSplitFoka</b>; |
| + | lanes: NEB prestained protein marker broad range, elution fraction 1, |
| + | elution fraction 2, elution fraction 3, elution fraction 4, elution |
| + | fraction 5, elution fraction 6, elution fraction 7, elution fraction 8, |
| + | elution fraction 9</small></td> |
| + | <td><small>SDS gel of protein expression and |
| + | purification with Streptag column of <b>pEx_DsbA_StrepDigSplitFoka</b>; |
| + | lanes: NEB prestained protein marker broad range, flow through fraction |
| + | 1, flow through fraction 2, flow through 3, flow through fraction 4, |
| + | washing fraction 1, washing fraction 2, washing fraction 3, unpurified |
| + | periplasm extract (frozen at -80°C)</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <br> |
| </div> | | </div> |
- | <div style="text-align: center;"><br /> | + | <ul> |
- | <br /> | + | <li>Plasmidpreparation </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>1 = |
| + | pEX-fos-split-fokA-K1<br> |
| + | 2 = pEX-fos-split-fokA-K2<br> |
| + | 3 = pMA-DsbA-strep-dig-split-fokA-K1<br> |
| + | 4 = pMA-DsbA-strep-dig-split-fokA-K2<br> |
| + | 5 = pMA-DsbA-his-dig-split-fokA-K1<br> |
| + | 6 = pMA-DsbA-his-dig-split-fokA-K2<br> |
| + | 7 = pMA-fos-split-fokA-K1<br> |
| + | 8 = pMA-fos-split-fokA-K2<br> |
| + | 9 = pBAD-fokA-K1<br> |
| + | 10= pBAD-fokA-K2<br> |
| + | 11 = pBAD-CAT-K1<br> |
| + | 12= pBAD-CAT-K2<br> |
| + | 13= pMA-shortlinker-fokA<br> |
| + | 14= pMA-longlinkerFoka<br> |
| + | 15= pMA-strep-dig<br> |
| + | 16= pMA-middlelinker-fokA<br> |
| + | 18=pMA-split-fokA<br> |
| + | ->sequencing of clones 1 and 2 of pBAD_Foka</small><br> |
| </div> | | </div> |
| + | <ul> |
| + | <li>Pellets of: </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>19=pMA-fokA <br> |
| + | 20=pEX-DsbA-strep-dig-split-fokA <br> |
| + | 21=pEX-strep-fluA(3.10) </small><br> |
| </div> | | </div> |
- | <div class="cleared"></div> | + | <ul> |
| + | <li>digest1: |
| + | - pBAD-iGEM(Midi 11.10)</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | pMA-DsbA-his-dig-split-fokA (Plasmidprep 14.10 Nr5)<br> |
| + | - pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4)<br> |
| + | </small></div> |
| + | <small><br> |
| + | </small> |
| + | <div style="margin-left: 40px;"><small>-10µlDNA<br> |
| + | -3µl buffer3<br> |
| + | -0.5µlBSA(100fold)<br> |
| + | -14.5µl water |
| + | -1µl EcoRI<br> |
| + | -1µl PstI<br> |
| + | - loaded on 1% agagel |
| + | </small><br> |
| </div> | | </div> |
| + | <ul> |
| + | <li>digest2: |
| + | - pMA-fos-split-fokA (Plasmidprep 14.10 Nr7)</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | pEX-fos-split-fokA-K1<br> |
| + | <br> |
| + | -10µlDNA<br> |
| + | -3µl buffer4<br> |
| + | -0.5µlBSA(100fold)<br> |
| + | -14.5µl water |
| + | -1µl xbaI<br> |
| + | -1µl NgoNIV</small> |
| + | <br> |
| + | <br> |
| </div> | | </div> |
| + | <div style="margin-left: 40px;"> |
| + | loaded on 1% agagel |
| + | <br> |
| </div> | | </div> |
- | <div class="art-Post"> | + | <ul> |
- | <div class="art-PostContent"><br /> | + | <li>digest3: |
- | <br /> | + | -pMA-DsbA-clone2 (5.10)</li> |
| + | </ul> |
| + | <br> |
| + | <div style="margin-left: 40px;"><small>-10µlDNA<br> |
| + | -3µl buffer4<br> |
| + | -0.5µlBSA(100fold)<br> |
| + | -14µl water |
| + | -1µl xbaI<br> |
| + | -1.5µl AgeI<br> |
| + | loaded on 3% agagel |
| + | *Gelextraction |
| + | -pBAD iGEM<br> |
| + | -DsbA-his-dig-split-foka<br> |
| + | -DsbA-strep-dig-split-foka<br> |
| + | -pMA-fos-dig-split-foka<br> |
| + | -pEX-fos-split-foka<br> |
| + | --> Picture</small><br> |
| </div> | | </div> |
- | <div class="art-Post-inner"> | + | <ul> |
- | <div class="cleared"></div> | + | <li>Ligation |
| + | -pBAD-DsbA-his-dig-split-fokA</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-pBAD-DsbA-strep-dig-split-fokA<br> |
| + | - pMA-fos-dig-split-fokA-DsbA<br> |
| + | -pEX-fos-split-fokA-DsbA<br> |
| + | -10µl Qick ligase buffer<br> |
| + | -1 µl Quick ligase<br> |
| + | -6µl Insert<br> |
| + | -3µl Vector</small><br> |
| </div> | | </div> |
| + | <ul> |
| + | <li> Transformation |
| + | of the ligation above in X1Blue competent cells</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;">-100µl and rest |
| + | of each transformation were plated on |
| + | Amp/Gluc plates |
| + | <br> |
| </div> | | </div> |
| + | <ul> |
| + | <li>made top agar (0.7% agar) |
| + | </li> |
| + | <li>nothing grew on IPTG/Amp plates for test in vivo assay |
| + | -> made control assays with</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | electroporating |
| + | cotransformed XL1Blue with ssM13DNA and plating them |
| + | directly to IPTG/Amp cells (no phage precipitation and infection of |
| + | ER2738) to test electrocompetence<br> |
| + | - infection of ER2738 with phages from lab phage stock and plating them |
| + | to IPTG/Amp cells to test plates<br> |
| + | ->put plates in 37° room overnight |
| + | </small><br> |
| </div> | | </div> |
| + | <ul> |
| + | <li>inoclutaion of ER2738 from lab stock and our stock in |
| + | LB+Tet |
| + | ->put them on shaker at 26°C </li> |
| + | <li> XL1 was el. transformed with the ligated |
| + | AGO-Phagmid library |
| + | (errore-prone PCR of the AGO-G3P construct into an phagmidvektor with |
| + | the Tor-A signalling sequence with and without amberstop from |
| + | yesterday) and plated everything (4 ml)on two big tubs. </li> |
| + | <li> new PCR for ssDNA from the digested pET39b+ |
| + | vector to get new target |
| + | for an additional AGO assay </li> |
| + | <li>prepaired ELISA to repeat yesterdays AGO-Phage-ELISA |
| + | tomorrow (because |
| + | yesterdays results were pretty contradictory -> we probably made |
| + | a |
| + | mistake somewhere) |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;"> 15.10.09, Laura, Caro, |
| + | Julia, Manu, Timo, Sarah, Hannes, Max, Anika, Christoph</h3> |
| + | <ul> |
| + | <li>ELISA:</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>-Blocking: |
| + | 350µl/well 1% BSA in TBST-EDTA, incubated at |
| + | 20°C for 2h gently shaking<br> |
| + | -5x washing with 200µl/well TBST-EDTA<br> |
| + | -Incubation of phages with oligos for 30min in 55°C waterbath<br> |
| + | -Incubation of 75µl/well phages for 1h at 20°C gently |
| + | shaking<br> |
| + | -5x washing with 200µl/well TBST-EDTA<br> |
| + | -Antibody: add 75µl/well of anti- M13 antibody (1:1000)<br> |
| + | -5x washing with 200µl/well TBST-EDTA<br> |
| + | -Substrate: add 75µl/well 1mg/ml ABTS; measure absorbance |
| + | immediately and after 3 and 5 min; 405nm</small><br> |
| + | </div> |
| + | <ul> |
| + | <li>made chemical competent XL1 Blue |
| + | *test digests of plasmid preparations from 14.10.09 |
| + | *seqencing of |
| + | - laura1:pExFosSplitFoka clone 2</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- |
| + | laura2:pMAFosSplitFoka clone 2<br> |
| + | - laura3:pMADsbAHisDigSplitFoka clone 2<br> |
| + | - laura4: pMADsbAStrepDigSplitFoka clone 1</small><br> |
| + | </div> |
| + | <ul> |
| + | <li>inoculation of - ER2738 from our stock</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- XL1 blue |
| + | from our stock<br> |
| + | -> in 50 ml LB+Tetracyclin each, put in 37°C |
| + | *inoculation of |
| + | -pBAD_Foka clone 1 and 2 from 14.10.09<br> |
| + | -pMAFoka clone 1from 04.10.09<br> |
| + | -pDsbA clone 2 from 05.10.09</small><br> |
| + | </div> |
| + | <ul> |
| + | <li>for in vivo assay |
| + | - measured pd.7.phage display library concentration new</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- mixed |
| + | 1:10^4, 2*10^6, 2*10^7, 2*10^8 dilutons with ER2738 (OD600 of |
| + | 0.46) and XL1 blue (OD600 of 0.54) each<br> |
| + | - let mixtures stand for 10min (infection time)<br> |
| + | - mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal |
| + | plates<br> |
| + | - put it in 37°C roomin the dark<br> |
| + | -pExStrepFluASplitFoki from 08.10.09</small><br> |
| + | </div> |
| + | <div style="margin-left: 40px;"><small>-> |
| + | in 5 ml LB+Ampicillin each, put in 37°C |
| + | </small><br> |
| + | </div> |
| + | <br> |
| + | <ul> |
| + | <li> new ssDNA production via pET39b+ digestion |
| + | product one-primer-pcr </li> |
| + | <li> elusions from the test panning was given to |
| + | xl1-> |
| + | infection-> |
| + | plated in tet cm -> cells infected with the phagmid should |
| + | grow->tomorrow picking clones and determination of the |
| + | phagmidinsert </li> |
| + | <li>new AGO BB pcr usind dmso, the gene cut out of the vektor |
| + | and only the |
| + | ad-on-tail-primers </li> |
| + | <li>starter cultures of AGO Aa and AGO Tt in BL21de3 in |
| + | DYT+Kan </li> |
| + | <li>starter cultures of pEx-His-Fos-Split-Foka in RV308 in |
| + | DYT+Amp+1% |
| + | Glucose |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;"> 16.10.09, Laura, Caro, |
| + | Julia, Manu, Timo, Sarah,Isabelle, Hannes, Max, Anika, Christoph</h3> |
| + | <ul> |
| + | <li>Made 10 L DYT</li> |
| + | <li>Inoculation of elutions from panning: E1#2 |
| + | (MnCl2+TBS)n°1-20, |
| + | E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20</li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;"> 17.10.09, Laura, Julia, |
| + | Manu, Hannes, Gerrit, Christoph</h3> |
| + | <ul> |
| + | <li>Expression of Ago Tt in Bl21de3 |
| + | OD measurement |
| + | </li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small><br> |
| + | </small></div> |
| + | <table |
| + | style="border-collapse: collapse; table-layout: fixed; width: 492pt; margin-left: 40px;" |
| + | border="1" cellpadding="0" cellspacing="0" |
| + | width="655"> |
| + | <tbody> |
| + | <tr> |
| + | <td class="xl15757" style="height: 15pt; width: 96pt;" |
| + | height="20" width="128"></td> |
| + | <td class="xl15757" style="width: 116pt;" |
| + | width="154"><small>1 hour |
| + | before</small></td> |
| + | <td class="xl15757" style="width: 88pt;" |
| + | width="117"></td> |
| + | <td class="xl15757" style="width: 48pt;" |
| + | width="64"></td> |
| + | <td class="xl15757" style="width: 48pt;" |
| + | width="64"></td> |
| + | <td class="xl15757" style="width: 48pt;" |
| + | width="64"></td> |
| + | <td class="xl15757" style="width: 48pt;" |
| + | width="64"></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"></td> |
| + | <td class="xl15757"><small>induction</small></td> |
| + | <td class="xl15757"><small>time |
| + | of induction</small></td> |
| + | <td class="xl15757"><small>1.5 |
| + | hours</small></td> |
| + | <td class="xl15757"><small>4 |
| + | hours</small></td> |
| + | <td class="xl15757"><small>6.5 |
| + | hours</small></td> |
| + | <td class="xl15757"><small>8 |
| + | hours</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"><small>flask 1</small></td> |
| + | <td class="xl15757" align="right"><small>0.29</small></td> |
| + | <td class="xl15757" align="right"><small>0.53</small></td> |
| + | <td class="xl15757" align="right"><small>0.86</small></td> |
| + | <td class="xl15757" align="right"><small>1.18</small></td> |
| + | <td class="xl15757" align="right"><small>1.32</small></td> |
| + | <td class="xl15757" align="right"><small>1.51</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"><small>flask 2</small></td> |
| + | <td class="xl15757" align="right"><small>0.31</small></td> |
| + | <td class="xl15757" align="right"><small>0.57</small></td> |
| + | <td class="xl15757" align="right"><small>0.91</small></td> |
| + | <td class="xl15757" align="right"><small>1.21</small></td> |
| + | <td class="xl15757" align="right"><small>1.38</small></td> |
| + | <td class="xl15757" align="right"><small>1.49</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"><small>flask 3</small></td> |
| + | <td class="xl15757" align="right"><small>0.32</small></td> |
| + | <td class="xl15757" align="right"><small>0.51</small></td> |
| + | <td class="xl15757" align="right"><small>0.9</small></td> |
| + | <td class="xl15757" align="right"><small>1.31</small></td> |
| + | <td class="xl15757" align="right"><small>1.32</small></td> |
| + | <td class="xl15757" align="right"><small>1.53</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"><small>flask 4</small></td> |
| + | <td class="xl15757" align="right"><small>0.28</small></td> |
| + | <td class="xl15757" align="right"><small>0.49</small></td> |
| + | <td class="xl15757" align="right"><small>0.88</small></td> |
| + | <td class="xl15757" align="right"><small>1.15</small></td> |
| + | <td class="xl15757" align="right"><small>1.33</small></td> |
| + | <td class="xl15757" align="right"><small>1.57</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"><small>flask 5</small></td> |
| + | <td class="xl15757" align="right"><small>0.27</small></td> |
| + | <td class="xl15757" align="right"><small>0.51</small></td> |
| + | <td class="xl15757" align="right"><small>0.89</small></td> |
| + | <td class="xl15757" align="right"><small>1.26</small></td> |
| + | <td class="xl15757" align="right"><small>1.32</small></td> |
| + | <td class="xl15757" align="right"><small>1.48</small></td> |
| + | </tr> |
| + | <tr style="height: 15pt;" height="20"> |
| + | <td class="xl15757" style="height: 15pt;" |
| + | height="20"><small>flask 6</small></td> |
| + | <td class="xl15757" align="right"><small>0.32</small></td> |
| + | <td class="xl15757" align="right"><small>0.51</small></td> |
| + | <td class="xl15757" align="right"><small>0.83</small></td> |
| + | <td class="xl15757" align="right"><small>1.24</small></td> |
| + | <td class="xl15757" align="right"><small>1.5</small></td> |
| + | <td class="xl15757" align="right"><small>1.61</small></td> |
| + | </tr> |
| + | <tr style="display: none;" height="0"> |
| + | <td style="width: 96pt;" width="128"></td> |
| + | <td style="width: 116pt;" width="154"></td> |
| + | <td style="width: 88pt;" width="117"></td> |
| + | <td style="width: 48pt;" width="64"></td> |
| + | <td style="width: 48pt;" width="64"></td> |
| + | <td style="width: 48pt;" width="64"></td> |
| + | <td style="width: 48pt;" width="64"></td> |
| + | </tr> |
| + | <small> |
| + | </small> |
| + | </tbody> |
| + | </table> |
| + | <small><br> |
| + | </small><br> |
| + | <ul> |
| + | <li>Dilution from XL1 from OD600 2,8 to OD600 0.07 |
| + | *Minipreps from : E1#2 (MnCl2+TBS)n°1-20, E2#1 |
| + | (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20 |
| + | wich are phagemids we cultured from elutions of panning from 13.10.09</li> |
| + | <li>IPTG/X-Gal |
| + | plates with infected ER2738 was blue, with the infected XL1 blue no |
| + | blue plaques could be seen -> so infection by phages was |
| + | positive |
| + | and lots of blue plaques were built up</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"> |
| + | <table style="text-align: left; width: 412px; height: 240px;" |
| + | border="1" cellpadding="2" cellspacing="0"> |
| + | <tbody> |
| + | <tr> |
| + | <td><a |
| + | href="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"><img |
| + | style="border: 0px solid ; width: 400px; height: 216px;" alt="" |
| + | src="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"></a><a |
| + | href="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG" |
| + | target="_blank"><wbr><wbr></a></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | </div> |
| + | <div style="margin-left: 40px;"> |
| + | <ul> |
| + | <li> made |
| + | new in vivo test assay with cotransformated, electrocompetent XL1 blue |
| + | - transformed M13 ss DNA from 17.08.09 into cells by electroporation at |
| + | 1.7kV</li> |
| + | </ul> |
| + | </div> |
| + | <div style="margin-left: 80px;"><small>- 1.5h |
| + | incubation time on 37°C shaker <br> |
| + | - 80, 10 and 5µl of electroporated cells were mixed with |
| + | 180µl XL1 blue and ER2738 with OD 600 of about 0.5<br> |
| + | - incubation time of 10 min at room temperature<br> |
| + | - mixing cells with 3ml topagar, vortexing shortly and plating on |
| + | IPTG/X-Gal plates<br> |
| + | - put in 37°C room overnight</small><br> |
| + | </div> |
| + | <ul> |
| + | <li>control of electrocompetence of XL1 blue by electroporation |
| + | of pET28a |
| + | with Kan resistance into XL1 blue and RV respecitvely |
| + | -> comparison in the end or if colonies will show up at all will |
| + | show if our cotransformated cells are competent at all |
| + | *inoculation of |
| + | - pMAFoka clone 1 from 04.10.09</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;"><small>- pMADsbA |
| + | clone 2 from 05.10.09<br> |
| + | - pBADFoka clone 1 from 15.10.09<br> |
| + | - pExStrepFluALongliFoki clone 1 from 08.10.09</small><br> |
| + | </div> |
| + | <ul> |
| + | <li>miniprep of all clones picked from the elutions of the test |
| + | panning to |
| + | determine the efficiency of the selection </li> |
| + | <li>new ssDNA via Pet39b+ one primer pcr </li> |
| + | <li>new infection of XL1 cells with the elution from the test |
| + | panning-> |
| + | this time plated on lb agar with tet cm and GLUC (to suppress the |
| + | expression of the phagmid insert) |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;"> 18.10.09 Manu</h3> |
| + | <ul> |
| + | <li>Expression of Ago Aa at 24°C |
| + | *Expression of GST-FosW at 30°C |
| + | </li> |
| + | </ul> |
| + | <h3 style="margin-left: 40px;"> 19.10.09, Laura, Caro, |
| + | Julia, Manu, Timo, Sarah, Isabel, Hannes, Max, Anika, Christoph</h3> |
| + | <ul> |
| + | <li> analysis of the geles of the digestion of the |
| + | clones picked from the |
| + | elution plates revealed not a single clone containing the ago |
| + | insert-> possibly the lack of gluc on the plate led to a |
| + | selection |
| + | against this insert-> new klones picked from the plate with |
| + | gluc </li> |
| + | <li>new ago cleavage assay reveald last saturdays promising |
| + | results as |
| + | false. it seems like it was just two conformations of one ssDNA |
| + | molecule </li> |
| + | <li>starter cultures of pEx-His-Fos-Split-Fok_a </li> |
| + | <li>protein purification of GST-FosW with Ni-NTA column |
| + | - French pressed, centrifuged 15min at 20000rpm, sonicated and filtered</li> |
| + | </ul> |
| + | <div style="margin-left: 40px;">- column was washed with |
| + | 25mM imidazole and eluted with 250mM imidazole |
| + | <br> |
| + | </div> |
| + | <ul> |
| + | <li> in vivo assay retried with shorter incubation |
| + | time |
| + | </li> |
| + | </ul> |
| + | <h3> 20.10.09 Manu, Laura, Anika, Julia, Timo, Sarah, Isabel, |
| + | Max, Gerrit, Christoph, Caro, Hannes</h3> |
| + | <ul> |
| + | <li> new round of expression with |
| + | pEx-His-Fos-Split-Fok_a in BL21de3at |
| + | 24°C </li> |
| + | </ul> |
| + | <table x:str="" |
| + | style="border-collapse: collapse; table-layout: fixed; width: 344pt;" |
| + | border="1" cellpadding="0" cellspacing="0" |
| + | width="459"> |
| + | <tbody> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" |
| + | style="height: 12.75pt; width: 96pt;" height="17" |
| + | width="128"><small>OD measurement</small></td> |
| + | <td class="xl1530995" style="width: 56pt;" |
| + | width="75"><small>time of</small></td> |
| + | <td class="xl1530995" style="width: 48pt;" |
| + | width="64"><small>2 hours</small></td> |
| + | <td class="xl1530995" style="width: 48pt;" |
| + | width="64"><small>3 hours</small></td> |
| + | <td class="xl1530995" style="width: 48pt;" |
| + | width="64"><small>3 hours</small></td> |
| + | <td class="xl1530995" style="width: 48pt;" |
| + | width="64"><small>4 hours</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small></small></td> |
| + | <td class="xl1530995"><small>induction</small></td> |
| + | <td class="xl1530995"><small></small></td> |
| + | <td class="xl1530995"><small></small></td> |
| + | <td class="xl1530995"><small>45 min</small></td> |
| + | <td class="xl1530995"><small>15 min</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small>flask 1</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>1.3</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>2.63</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.47</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.29</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.99</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small>flask 2</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>1.19</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>2.87</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.87</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.51</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.62</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small>flask 3</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>1.5</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.22</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.45</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>5.03</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>5.41</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small>flask 4</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>1.03</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>2.9</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.21</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>5.11</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>5.88</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small>flask 5</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>0.81</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>2.81</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.76</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.29</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>4.76</small></td> |
| + | </tr> |
| + | <tr style="height: 12.75pt;" height="17"> |
| + | <td class="xl1530995" style="height: 12.75pt;" |
| + | height="17"><small>flask 6</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>1.52</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>2.72</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.71</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>3.95</small></td> |
| + | <td class="xl1530995" x:num="" align="right"><small>5.17</small></td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | |
| + | <div class="art-Footer-inner"> |
| + | <div class="art-Footer-text"> |
| + | <p>contact: <a |
| + | href="mailto:freigem09@googlemail.com">freigem09@googlemail.com</a><br /> |
| + | </p> |
| + | </div> |
| + | </div> |
| + | <div class="art-Footer-background"></div> |
| </div> | | </div> |
- | <br />
| |
| </div> | | </div> |
| </div> | | </div> |
- | <div class="cleared"></div>
| |
| </div> | | </div> |
| </body> | | </body> |
| </html> | | </html> |