Team:Freiburg bioware/Notebook/October

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<h2 class="art-PostHeaderIcon-wrapper">October<span
+
<h2 class="art-PostHeaderIcon-wrapper"> &nbsp;<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>01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph,
 +
Caro, Julia, Anika, Isabel</h3>
<br />
<br />
-
<div style="text-align: left;">
+
<ul>
-
 
+
  <li>&nbsp;Tetracycline; 10 x 1ml, Concentration: 25 g/l,
-
<h3>01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel</h3>
+
stored in -20&deg;C, box with antibiotics
-
<br>* Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
+
  </li>
-
<br>* Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
+
  <li>&nbsp;Chloramphenicol; 10 x 1ml, Concentration: 25 g/l,
-
<br>* plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
+
stored in -20&deg;C, box with antibiotics
-
<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>&nbsp;plasmid preparation of
-
<br>
+
pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
-
<br>* protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column
+
  </li>
-
<br>* SDS gel of protein purification HisFluASplitFoki
+
  <li>&nbsp;plasmid preparation of
-
 
+
pEX-Strep-Dig-Split-Fok(active) Klon 1
-
[[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>&nbsp;glycerin stock of
-
<br>* pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4)
+
pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
-
<br>*phage display:
+
(RV308), stored at -80&deg;C
-
- desalt ligation products (for electroporation)<br>
+
  </li>
-
pool samples (~80µl 445+87/89 and ~52µl 445+87/88)<br>
+
</ul>
-
add 1 volume of isopropanol, mix<br>
+
<br />
-
-80°C for 10 minutes<br>
+
<ul>
-
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant<br>
+
  <li>protein purification of HisFluASplitFoki (expressed in
-
add 1 volume of 75% ethanol (without mixing)<br>
+
BL21de3) with NiNTA column
-
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant<br>
+
  </li>
-
let dry on heat block (50°C), lid open<br>
+
  <li>SDS gel of protein purification HisFluASplitFoki
-
add 25µl water<br>
+
[[Image:Freiburg09_011009_pg_HisFluASplitFoki011.jpg|none|thumb|SDS-Gel;
-
thermo shaker for 1h, 45°C, 1000rpm
+
HisFluASplitFoki; Lanes: NEB prestained protein marker, elution
-
 
+
fraction 1, elution fraction 2, elution fraction 3, elution fraction 4,
-
- PCR (4 samples each template):<br>
+
elution fraction 5, elution fraction 6, elution fraction 7, flow
-
buffer (with MgCl2): 5µl<br>
+
through fraction 2, washing fraction 2|400x400px]]
-
primer #7: 1,5µl<br>
+
  </li>
-
primer #1: 1,17µl<br>
+
  <li>pooled fraction 2-5, dialysis over night in dialysis buffer
-
dNTPs: 2µl<br>
+
(30mM NaCl, 20mM Tris-HCl, pH 7.4)
-
Taq: 1µl<br>
+
  </li>
-
MnCl2: 0,5µl<br>
+
  <li>phage display:
-
MgCl2: 5µl<br>
+
- desalt ligation products (for electroporation)</li>
-
water: 33µl<br>
+
</ul>
-
DNA (425 or 428): 5µl<br>
+
<div style="margin-left: 40px;">pool samples
-
 
+
(~80&micro;l 445+87/89 and ~52&micro;l 445+87/88)<br />
-
- PCR:<br>
+
add 1 volume of isopropanol, mix<br />
-
DNA templates: pJs#448 (0,3µl), pJs#449 (0,3µl), pJs#375 (1µl), pJs#413 (0,3µl), pJs#445 (0,3µl)<br>
+
-80&deg;C for 10 minutes<br />
-
primers (#95, #7): 1,5µl<br>
+
centrifuge for 10 minutes at max rpm, 4&deg;C; discard supernatant<br />
-
high fidelity buffer: 5µl<br>
+
add 1 volume of 75% ethanol (without mixing)<br />
-
dNTPs: 1µl<br>
+
centrifuge for 10 minutes at max rpm, 4&deg;C; discard supernatant<br />
-
TMenzyme: 0,3µl<br>
+
let dry on heat block (50&deg;C), lid open<br />
-
water: 39,7µl<br>
+
add 25&micro;l water<br />
-
<br>* Overnight Culture RV308 for Competent Cells, on shaker in 37 °C room
+
thermo shaker for 1h, 45&deg;C, 1000rpm
-
<br>*analysis of sequences from 28.09.09
+
- PCR (4 samples each template):<br />
-
<br>*digest of pExStrepDigSplitFoka prep from 30.09.09
+
buffer (with MgCl2): 5&micro;l<br />
-
Plasmid: 15 µl<br>
+
primer #7: 1,5&micro;l<br />
-
water: 9 µl<br>
+
primer #1: 1,17&micro;l<br />
-
BSA: 0.5 µl NEB iGEM stock<br>
+
dNTPs: 2&micro;l<br />
-
buffer: 3 µl buffer 3 NEB iGEM stock<br>
+
Taq: 1&micro;l<br />
-
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br>
+
MnCl2: 0,5&micro;l<br />
-
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br>
+
MgCl2: 5&micro;l<br />
-
<br>*digest of PCR product RBSStrepDigSplitFoka from 30.09.09
+
water: 33&micro;l<br />
-
Plasmid: 10 µl <br>
+
DNA (425 or 428): 5&micro;l<br />
-
water: 14 µl<br>
+
- PCR:<br />
-
BSA: 0.5 µl NEB iGEM stock<br>
+
DNA templates: pJs#448 (0,3&micro;l), pJs#449 (0,3&micro;l),
-
buffer: 3 µl buffer 3 NEB iGEM stock<br>
+
pJs#375 (1&micro;l), pJs#413 (0,3&micro;l), pJs#445
-
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br>
+
(0,3&micro;l)<br />
-
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br>
+
primers (#95, #7): 1,5&micro;l<br />
-
<br>*digest of pExHisFluaSplitFoki prep from 24.08.09
+
high fidelity buffer: 5&micro;l<br />
-
Plasmid: 15 µl <br>
+
dNTPs: 1&micro;l<br />
-
water: 9 µl<br>
+
TMenzyme: 0,3&micro;l<br />
-
BSA: 0.5 µl NEB iGEM stock<br>
+
water: 39,7&micro;l<br />
-
buffer: 3 µl buffer 2 NEB iGEM stock<br>
+
</div>
-
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br>
+
<br />
-
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br>
+
<ul>
-
-> put in 37°C room for 2h
+
  <li>&nbsp;Overnight Culture RV308 for Competent Cells, on
-
->preparative gel<br>
+
shaker in 37 &deg;C room
-
[[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]]
+
  </li>
-
interpretation: bands had right size but pExStrepDigSplitFoka showed unexpectedly low concentration<br>
+
  <li>analysis of sequences from 28.09.09
-
->gelextraction <br>
+
  </li>
-
ligation of<br>
+
  <li>digest of pExStrepDigSplitFoka prep from 30.09.09
-
- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)<br>
+
Plasmid: 15 &micro;l</li>
-
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br>
+
</ul>
-
<br>*Inoculation of  
+
<div style="margin-left: 40px;">water: 9 &micro;l<br />
-
- pJS419_StrepDigSplitFoka<br>
+
BSA: 0.5 &micro;l NEB iGEM stock<br />
-
- pJS419_HisDigSplitFoka<br>
+
buffer: 3 &micro;l buffer 3 NEB iGEM stock<br />
-
- pEx_HisFluA<br>
+
Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock<br />
-
- pEx_HisDigMiddleLiFoka<br>
+
Restriction_enzyme_2 : 1 &micro;l PstI from NEB KuKlabstock <br />
-
<br>*digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
+
</div>
-
<br>*Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09
+
<br />
-
--> make glycerol stocks tomorrow
+
<ul>
-
<br>*agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
+
  <li>digest of PCR product RBSStrepDigSplitFoka from 30.09.09
-
<br>*cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more)
+
Plasmid: 10 &micro;l </li>
-
<br>*gel slices stored at -4°C over night
+
</ul>
-
 
+
<div style="margin-left: 40px;">water: 14 &micro;l<br />
-
<h3>02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max</h3>
+
BSA: 0.5 &micro;l NEB iGEM stock<br />
-
<br>* phage display
+
buffer: 3 &micro;l buffer 3 NEB iGEM stock<br />
-
- OD600 of the pre-culture should be 0,2 <br>
+
Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock<br />
-
- 37°C shaker<br>
+
Restriction_enzyme_2 : 1 &micro;l PstI from NEB KuKlabstock <br />
-
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br>
+
</div>
-
- Apportion culture into 10x50 ml Falkon tubes<br>
+
<br />
-
- 15 minutes on ice<br>
+
<div style="margin-left: 40px;">*digest of
-
- Centrifuge 10 minutes at 4°C / 2500 g<br>
+
pExHisFluaSplitFoki prep from 24.08.09
-
- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)<br>
+
Plasmid: 15 &micro;l <br />
-
- resuspend pellets in 25 ml H2O (10-> 8 tubes)<br>
+
</div>
-
- 15 minutes on ice<br>
+
<div style="margin-left: 40px;">water: 9 &micro;l<br />
-
- Centrifuge 10 minutes at 4°C / 2500 g<br>
+
BSA: 0.5 &micro;l NEB iGEM stock<br />
-
- Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4 tubes)<br>
+
buffer: 3 &micro;l buffer 2 NEB iGEM stock<br />
-
- 15 minutes on ice<br>
+
Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock<br />
-
- Centrifuge 10 min at 4°C / 2500 <br>
+
Restriction_enzyme_2 : 1 &micro;l SpeI from NEB KuKlabstock <br />
-
- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-> 2 tubes)<br>
+
-&gt; put in 37&deg;C room for 2h
-
- 5 minutes on ice<br>
+
-&gt;preparative gel<br />
-
- Centrifuge 10 minutes at 4°C / 2500 g<br>
+
<br />
-
- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)<br>
+
<table style="text-align: left; width: 623px; height: 529px;"
-
- 5 minutes on ice<br>
+
border="0" cellpadding="0" cellspacing="0">
-
- Centrifuge 10 min at 4°C / 2500 g<br>
+
  <tbody>
-
- Discard supernatant, pool pellets in 1 ml 10% DMSO<br>
+
    <tr>
-
- Measure OD600 (1:100 dilution), OD should be 0,4<br>
+
      <td><img alt=""
-
- Make aliquots à 80 µl, souse with N2<br>
+
src="https://static.igem.org/mediawiki/2009/3/3b/Freiburg09_011009_fokverdaue.JPG" /></td>
-
- Store at -80°C<br>
+
    </tr>
-
<br>*glycerol stock of pEx_HisFluASplitFoki in BL21de3
+
    <tr>
-
<br>*SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09
+
      <td>Agarose gel; Lanes: 1.Gene ruler ladder mix of
-
--> do a Western Blot
+
fermentas,
-
 
+
2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka</td>
-
<br>*new ampicilin 100 aliquots  
+
    </tr>
-
<br>*preparation of M13dsDNA  
+
  </tbody>
-
<br>*chem. competent cells aliqots
+
</table>
-
 
+
<br />
-
 
+
<br />
-
<br>*Periplasma Project
+
</div>
-
 
+
<div style="margin-left: 40px;">&nbsp;interpretation:
-
Digestion:<br>
+
bands had right size but pExStrepDigSplitFoka showed
-
 
+
unexpectedly low concentration<br />
-
- digested with xba and spe
+
-&gt;gelextraction <br />
-
--> GelBilder
+
ligation of<br />
-
 
+
- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)<br />
-
<br>* Gelextraction
+
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br />
-
 
+
</div>
-
Min Elute Gel Extraction Kit<br>
+
<br />
-
- mesure mass: StrepFokA = 50 mg<be>
+
<ul>
-
- JS418= 90mg<br>
+
  <li>Inoculation of - pJS419_StrepDigSplitFoka</li>
-
-JS419=80mg<br>
+
</ul>
-
- HisFokA = 250mg<br>
+
<div style="margin-left: 40px;">- pJS419_HisDigSplitFoka<br />
-
 
+
- pEx_HisFluA<br />
-
Nandropdata<br>
+
- pEx_HisDigMiddleLiFoka
-
 
+
<br />
-
<br>*Ligation
+
</div>
 +
<ul>
 +
  <li>digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and
 +
pMA_scFa-anti_NIP for recloning
 +
  </li>
 +
  <li>Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony
 +
of transformation from 28.09.09
 +
--&gt; make glycerol stocks tomorrow
 +
  </li>
 +
  <li>agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a,
 +
pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
 +
  </li>
 +
  <li>cut out insert bands (digestion of pEX didn't work, because
 +
there's no EcoRI recognition site any more)
 +
  </li>
 +
  <li>gel slices stored at -4&deg;C over night
 +
  </li>
 +
</ul>
 +
<h3>02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell,
 +
Sarah, Caro, Timo, Christoph, Max</h3>
 +
<br />
 +
<ul>
 +
  <li>phage display
 +
- OD600 of the pre-culture should be 0,2 </li>
 +
</ul>
 +
<div style="margin-left: 40px;">- 37&deg;C shaker<br />
 +
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br />
 +
- Apportion culture into 10x50 ml Falkon tubes<br />
 +
- 15 minutes on ice<br />
 +
- Centrifuge 10 minutes at 4&deg;C / 2500 g<br />
 +
- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)<br />
 +
- resuspend pellets in 25 ml H2O (10-&gt; 8 tubes)<br />
 +
- 15 minutes on ice<br />
 +
- Centrifuge 10 minutes at 4&deg;C / 2500 g<br />
 +
- Discard supernatant, resuspend pellets in 25 ml H2O (8-&gt; 4
 +
tubes)<br />
 +
- 15 minutes on ice<br />
 +
- Centrifuge 10 min at 4&deg;C / 2500 <br />
 +
- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-&gt;
 +
2 tubes)<br />
 +
- 5 minutes on ice<br />
 +
- Centrifuge 10 minutes at 4&deg;C / 2500 g<br />
 +
- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)<br />
 +
- 5 minutes on ice<br />
 +
- Centrifuge 10 min at 4&deg;C / 2500 g<br />
 +
- Discard supernatant, pool pellets in 1 ml 10% DMSO<br />
 +
- Measure OD600 (1:100 dilution), OD should be 0,4<br />
 +
- Make aliquots &agrave; 80 &micro;l, souse with N2<br />
 +
- Store at -80&deg;C<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>glycerol stock of pEx_HisFluASplitFoki in BL21de3
 +
  </li>
 +
  <li>SDS gel of pool (elution fraction 2-5) from protein
 +
expression (HisFluASplitFoki) from 01.10.09
 +
--&gt; do a Western Blot
 +
  </li>
 +
  <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;">- digested with xba and
 +
spe --&gt; GelBilder
 +
<br />
 +
<br />
 +
</div>
 +
<ul>
 +
  <li>&nbsp;Gelextraction
 +
Min Elute Gel Extraction Kit</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- 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><br />
 +
<be></be></div>
 +
<be><br />
 +
</be>
 +
<ul>
 +
  <li><be>Ligation
- with Quickligase
- with Quickligase
-
 
+
JS119-StrepFokA</be></li>
-
JS119-StrepFokA<br>
+
</ul>
-
JS118-Strep<br>
+
<div style="margin-left: 40px;"><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&deg;C
-
- 15 min at 25°C
+
</be><br />
-
 
+
<be></be></div>
-
<br>*Transformation
+
<ul>
-
- 2YT-Medium 950 µL to 50µl cells and DNA <br>
+
  <li><be>Transformation
-
 
+
- 2YT-Medium 950 &micro;L to 50&micro;l cells and DNA </be></li>
-
BL21-JS190<br>
+
</ul>
-
BL21-JS119StrepFokA<br>
+
<div style="margin-left: 40px;"><be>BL21-JS190</be><br />
-
BL21-JS118Strep<br>
+
<be>BL21-JS119StrepFokA</be><br />
-
BL21-JS118HisFokA<br>
+
<be>BL21-JS118Strep</be><br />
-
BL21-JS19HisFokA
+
<be>BL21-JS118HisFokA</be><br />
-
 
+
<be>BL21-JS19HisFokA
-
- Plated on KM plats<br>
+
- Plated on KM plats</be><br />
-
 
+
<be></be></div>
-
<br>* Preparation of overnight cultures 5 ml LB medium each
+
<be><br />
-
- pBad+Kan<br>
+
</be>
-
-pET39B(+)<br>*Amp<br>
+
<ul>
-
--> Stored in 37°C room
+
  <li><be>&nbsp;Preparation of overnight cultures 5 ml
-
 
+
LB medium each
-
<br>*Digest:
+
- pBad+Kan</be></li>
-
- pMASplitFoka<br>
+
</ul>
-
- pMAFoka<br>
+
<div style="margin-left: 40px;"><be>-pET39B(+)</be><br />
-
- pMAFoki<br>
+
<be></be></div>
-
- pMASplitFoki<br>
+
<ul>
-
- pMALongLiFoka<br>
+
  <li><be>Amp</be></li>
-
- pMALongliFoki<br>
+
</ul>
-
- pMAShortli<br>
+
<div style="margin-left: 40px;"><be>--&gt;
-
- pMAMiddleli<br>
+
Stored in 37&deg;C room
-
- pMALongli<br>
+
</be><br />
-
- pExStrepFluA<br>
+
<be></be></div>
-
 
+
<ul>
-
 
+
  <li><be>Digest:
-
<br>*preparative gel
+
- pMASplitFoka</be></li>
-
[[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]]
+
</ul>
-
->interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly
+
<div style="margin-left: 40px;"><be>- pMAFoka</be><br />
-
<br>*gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09
+
<be>- pMAFoki</be><br />
-
 
+
<be>- pMASplitFoki</be><br />
-
 
+
<be>- pMALongLiFoka</be><br />
-
<br>* digest and ligation into new pMA:<br>
+
<be>- pMALongliFoki</be><br />
-
-pMASplitFokA clone1 19.08.09<br>
+
<be>- pMAShortli</be><br />
-
-pMAFokA clone2 05.08.09<br>
+
<be>- pMAMiddleli</be><br />
-
-pMAFoki clone2 05.08.09<br>
+
<be>- pMALongli</be><br />
-
-pMASplitFoki clone2 06.08.09<br>
+
<be>- pExStrepFluA</be><br />
-
-pMAshortLi clone2 10.09.09<br>
+
<be></be></div>
-
-pMAmiddelLi clone2 10.09.09<br>
+
<be><br />
-
-pMAlongLi clone1 10.09.09<br>
+
</be>
-
-pMAlongLiFokA clone1 10.09.09<br>
+
<ul>
-
-pMAlongLiFoki clone1 10.09.09<br>
+
  <li><be>preparative gel
-
digestion with EcoRI and SpeI (vector and insert)
+
[[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel;
-
<br>
+
Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki,
-
<br>* Transformation of:<br>
+
5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli,
-
-pMAFokA clone2 05.08.09<br>  
+
9.pMALongli, 10.pExStrepFluA&lt;|400x400px]]
-
-pMAFoki clone2 05.08.09<br>
+
-&gt;interpretation: just the linker resulted in too short
-
-pMASplitFoki clone2 06.08.09<br>
+
fragments,
-
 
+
thus the hybridized linkers have to be cloned in pMA directly
-
<br>*plasmidprep of:<br>
+
    </be></li>
-
1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick<br>
+
  <li><be>gelextraction of digest from 02.10.09 and also
-
2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick<br>
+
pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from
-
3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick<br><br>
+
01.10.09
-
 
+
    </be></li>
-
4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick <br>
+
  <li><be>digest and ligation into new pMA:</be></li>
-
5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick<br>
+
</ul>
-
6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick<br><br>
+
<div style="margin-left: 40px;"><be>-pMASplitFokA
-
 
+
clone1 19.08.09</be><br />
-
7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit1<br>
+
<be>-pMAFokA clone2 05.08.09</be><br />
-
8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit2<br>
+
<be>-pMAFoki clone2 05.08.09</be><br />
-
9. pEX_DsbA+Strep+Dig+Split+FokA Clone3<br><br>
+
<be>-pMASplitFoki clone2 06.08.09</be><br />
-
 
+
<be>-pMAshortLi clone2 10.09.09</be><br />
-
10. pEX_DsbA+His+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit3<br>
+
<be>-pMAmiddelLi clone2 10.09.09</be><br />
-
11. pEX_DsbA+His+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit4<br>
+
<be>-pMAlongLi clone1 10.09.09</be><br />
-
12. pEX_DsbA+His+Dig+Split+FokA Clone3<br><br>
+
<be>-pMAlongLiFokA clone1 10.09.09</be><br />
-
 
+
<be>-pMAlongLiFoki clone1 10.09.09</be><br />
-
<br>*testdigest of:
+
<be>digestion with EcoRI and SpeI (vector and insert)
-
V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick<br>
+
</be><br />
-
V2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick<br>
+
<be></be></div>
-
V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick<br><br>
+
<be><br />
-
 
+
</be>
-
V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick <br>
+
<ul>
-
V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick<br>
+
  <li><be>&nbsp;Transformation of:</be></li>
-
V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick<br><br>
+
</ul>
-
 
+
<div style="margin-left: 40px;"><be>-pMAFokA clone2
-
V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 <br>
+
05.08.09</be><br />
-
V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 <br>
+
<be> -pMAFoki clone2 05.08.09</be><br />
-
V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3<br><br>
+
<be></be></div>
-
 
+
<div style="margin-left: 40px;"><be>-pMASplitFoki
-
V10. pEX_DsbA+His+Dig+Split+FokA Clone1 <br>
+
clone2 06.08.09</be><br />
-
V11. pEX_DsbA+His+Dig+Split+FokA Clone2 <br>
+
<be></be></div>
-
V12. pEX_DsbA+His+Dig+Split+FokA Clone3<br><br>
+
<be><br />
-
 
+
</be>
-
V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)<br>
+
<ul>
-
V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)<br>
+
  <li><be>plasmidprep of:</be></li>
-
with NcoI-HF and XbaI 1h at 37°C<br>
+
</ul>
-
 
+
<div style="margin-left: 40px;"><be>1.
-
--> geldbild
+
pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be><br />
-
 
+
<be>2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br />
 +
<be>3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be><br />
 +
<be></be><br />
 +
<be>4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick </be><br />
 +
<be>5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick</be><br />
 +
<be>6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick</be><br />
 +
<be></be><br />
 +
<be>7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --&gt; seq
 +
02.10.09 Gerrit1</be><br />
 +
<be>8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --&gt; seq
 +
02.10.09 Gerrit2</be><br />
 +
<be>9. pEX_DsbA+Strep+Dig+Split+FokA Clone3</be><br />
 +
<be></be><br />
 +
<be>10. pEX_DsbA+His+Dig+Split+FokA Clone1 --&gt; seq
 +
02.10.09 Gerrit3</be><br />
 +
<be>11. pEX_DsbA+His+Dig+Split+FokA Clone2 --&gt; seq
 +
02.10.09 Gerrit4</be><br />
 +
<be>12. pEX_DsbA+His+Dig+Split+FokA Clone3</be><br />
 +
<be></be></div>
 +
<be><br />
 +
<br />
 +
</be>
 +
<ul>
 +
  <li><be>testdigest of:
 +
V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be></li>
 +
</ul>
 +
<div style="margin-left: 40px;"><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&deg;C</be><br />
 +
<be> --&gt; geldbild
 +
</be></div>
<h3>03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph </h3>
<h3>03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph </h3>
-
<br>*plasmid prep of pET and pBad and Glytsocks(Xbl)
+
<br />
-
<br>*new aliquots Ampicilin (70%EtOH)
+
<ul>
-
<br>*Test digest M13dsDNA+fokI as control for dsDNA , the pictures showed the two expected lanes(hardly visible on the printout).
+
  <li>plasmid prep of pET and pBad and Glytsocks(Xbl)
-
 
+
  </li>
-
[[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>new aliquots Ampicilin (70%EtOH)
-
<br>*digest of  
+
  </li>
-
- pEx-Strep-Dig-LongLinker-Fok(inactive)<br>
+
  <li>Test digest M13dsDNA+fokI as control for dsDNA , the
-
- pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br>
+
pictures
-
- pEx-Strep-Dig-ShortLinker-Fok(inactive)<br>
+
showed the two expected lanes(hardly visible on the printout).
-
- pEx-Strep-Dig-SplitLinker-Fok(inactive)<br>
+
[[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel;
-
- pEx-His-Dig-SplitLinker_Fok(inactive)<br>
+
M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09,
-
- pEx-His-Dig-SplitLinker_Fok(active)<br>
+
M13dsDNA02.10.09 digest fokI |400x400px]]
-
- pEx-Strep-Dig-SplitLinker-Fok(active)<br>
+
  </li>
-
- pEx-His-Dig-LongLinker-Fok(inactive)<br>
+
</ul>
-
- pEx-His-FluA-LongLinker-Fok(inactive)<br>
+
<ul>
-
- pEx-His-FluA-MiddleLinker-Fok(inactive)<br>
+
  <li>digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)</li>
-
- pEx-His-FluA-ShortLinker-Fok(inactive)<br>
+
</ul>
-
- pEx-His-FluA-SplitLinker-Fok(inactive)<br>
+
<div style="margin-left: 40px;">-
-
- pEx-His-Dig<br>
+
pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br />
-
- pMA<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
+
<br />
-
- pMA<br>
+
</div>
-
with XbaI and AgeI
+
<ul>
-
 
+
  <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>
-
- 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;">-
-
- 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
-
 
+
<br />
-
<br>*inoculation of:
+
</div>
-
-pMASplitFokA clone1 19.08.09<br>
+
<ul>
-
-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;">-pMAFokA clone2 05.08.09
-
-pMAmiddelLi clone2 10.09.09<br>
+
(new)<br />
-
-pMAlongLi clone1 10.09.09<br>
+
-pMAFoki clone2 05.08.09 (new)<br />
-
-pMAlongLiFokA clone1 10.09.09<br>
+
-pMASplitFoki clone2 06.08.09 (new)<br />
-
-pMAlongLiFoki clone1 10.09.09<br><br>
+
-pMAshortLi clone2 10.09.09<br />
-
-pMAFokA clone2 05.08.09 (old)<br>
+
-pMAmiddelLi clone2 10.09.09<br />
-
-pMAFoki clone2 05.08.09 (old)<br>
+
-pMAlongLi clone1 10.09.09<br />
-
-pMASplitFoki clone2 06.08.09 (old)<br>
+
-pMAlongLiFokA clone1 10.09.09<br />
-
 
+
-pMAlongLiFoki clone1 10.09.09<br />
-
<br>*inoculation and plasmid preperation:
+
<br />
 +
-pMAFokA clone2 05.08.09 (old)<br />
 +
-pMAFoki clone2 05.08.09 (old)<br />
 +
-pMASplitFoki clone2 06.08.09 (old)<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <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>
 +
  <li>BB Ago Pcr via Taq
 +
  </li>
 +
  <li>Started makin new VCS M13 Phages see Protocol day 1
 +
  </li>
 +
</ul>
<h3>Plan for 04.10.09</h3>
<h3>Plan for 04.10.09</h3>
-
<br>*inoculation of
+
<br />
-
- pMA-Strep-Dig-LongLinker-Fok(inactive)<br>
+
<ul>
-
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br>
+
  <li>inoculation of
-
- pMA-Strep-Dig-ShortLinker-Fok(inactive)<br>
+
- pMA-Strep-Dig-LongLinker-Fok(inactive)</li>
-
- pMA-Strep-Dig-SplitLinker-Fok(inactive)<br>
+
</ul>
-
- pMA-His-Dig-SplitLinker_Fok(inactive)<br>
+
<div style="margin-left: 40px;">-
-
- pMA-His-Dig-SplitLinker_Fok(inactive)<br>
+
pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br />
-
- pMA-Strep-Dig-SplitLinker-Fok(active)<br>
+
- pMA-Strep-Dig-ShortLinker-Fok(inactive)<br />
-
- pMA-His-Dig-LongLinker-Fok(inactive)<br>
+
- pMA-Strep-Dig-SplitLinker-Fok(inactive)<br />
-
- pMA-His-FluA-LongLinker-Fok(inactive)<br>
+
- pMA-His-Dig-SplitLinker_Fok(inactive)<br />
-
- pMA-His-FluA-MiddleLinker-Fok(inactive)<br>
+
- pMA-His-Dig-SplitLinker_Fok(inactive)<br />
-
- pMA-His-FluA-ShortLinker-Fok(inactive)<br>
+
- pMA-Strep-Dig-SplitLinker-Fok(active)<br />
-
- pMA-His-FluA-SplitLinker-Fok(inactive)<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
+
<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>
-
<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;">-pMAFokA <br />
-
-pMAFokA (old)<br>
+
-pMAFoki <br />
-
-pMAFoki (old)<br>
+
-pMASplitFoki <br />
-
-pMASplitFoki (old)<br>
+
-pMAshortLi<br />
-
 
+
-pMAmiddelLi<br />
 +
-pMAlongLi <br />
 +
-pMAlongLiFokA <br />
 +
-pMAlongLiFoki <br />
 +
-pMAFokA (old)<br />
 +
-pMAFoki (old)<br />
 +
-pMASplitFoki (old)<br />
 +
</div>
<h3>04.10.09, Laura, Anika, Max</h3>
<h3>04.10.09, Laura, Anika, Max</h3>
-
<br>* Plasmid preparation of  
+
<br />
-
-pMASplitFokA <br>
+
<ul>
-
-pMAFokA <br>
+
  <li>&nbsp;Plasmid preparation of -pMASplitFokA </li>
-
-pMAFoki <br>
+
</ul>
-
-pMASplitFoki <br>
+
<div style="margin-left: 40px;">-pMAFokA <br />
-
-pMAshortLiFokA<br>
+
-pMAFoki <br />
-
-pMAshortLiFoki<br>
+
-pMASplitFoki <br />
-
-pMAmiddelLiFokA<br>
+
-pMAshortLiFokA<br />
-
-pMAmiddelLiFoki<br>
+
-pMAshortLiFoki<br />
-
-pMAlongLiFokA <br>
+
-pMAmiddelLiFokA<br />
-
-pMAlongLiFoki <br>
+
-pMAmiddelLiFoki<br />
-
-pMAFokA (old)<br>
+
-pMAlongLiFokA <br />
-
-pMAFoki (old)<br>
+
-pMAlongLiFoki <br />
-
-pMASplitFoki (old)<br>
+
-pMAFokA (old)<br />
-
clone 1 and 2,respectively - results see notebook<br>
+
-pMAFoki (old)<br />
-
<br>* Glycerolstock of <br>
+
-pMASplitFoki (old)<br />
-
-pMASplitFokA <br>
+
clone 1 and 2,respectively - results see notebook
-
-pMAFokA <br>
+
<br />
-
-pMAFoki <br>
+
</div>
-
-pMASplitFoki <br>
+
<ul>
-
-pMAshortLiFokA<br>
+
  <li>&nbsp;Glycerolstock of </li>
-
-pMAshortLiFoki<br>
+
</ul>
-
-pMAmiddelLiFokA<br>
+
<div style="margin-left: 40px;">-pMASplitFokA <br />
-
-pMAmiddelLiFoki<br>
+
-pMAFokA <br />
-
-pMAlongLiFokA <br>
+
-pMAFoki <br />
-
-pMAlongLiFoki <br>
+
-pMASplitFoki <br />
-
-pMAFokA (old)<br>
+
-pMAshortLiFokA<br />
-
-pMAFoki (old)<br>
+
-pMAshortLiFoki<br />
-
-pMASplitFoki (old)<br>
+
-pMAmiddelLiFokA<br />
 +
-pMAmiddelLiFoki<br />
 +
-pMAlongLiFokA <br />
 +
-pMAlongLiFoki <br />
 +
-pMAFokA (old)<br />
 +
-pMAFoki (old)<br />
 +
-pMASplitFoki (old)<br />
clone 3
clone 3
-
<br>* Pellets of<br>
+
<br />
-
-pMASplitFokA <br>
+
</div>
-
-pMAFokA <br>
+
<ul>
-
-pMAFoki <br>
+
  <li>&nbsp;Pellets of</li>
-
-pMASplitFoki <br>
+
</ul>
-
-pMAshortLiFokA<br>
+
<div style="margin-left: 40px;">-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 - stored in Pelletbox, -20°C
+
-pMAlongLiFoki <br />
-
<br>*Digest of  
+
-pMAFokA (old)<br />
-
1. pEXHisDigMiddleLFoki
+
-pMAFoki (old)<br />
 +
-pMASplitFoki (old)<br />
 +
</div>
 +
<div style="margin-left: 40px;">clone 1 and 2,respectively
 +
- stored in Pelletbox, -20&deg;C
 +
<br />
 +
</div>
 +
<ul>
 +
  <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;">5. pMAShortLFoki<br />
-
7. pMAMiddleLFoki<br>
+
6. pMAMiddleLFoka<br />
-
8. pMALongLFoka<br>
+
7. pMAMiddleLFoki<br />
-
9. pMALongLFoki<br>
+
8. pMALongLFoka<br />
-
10.pEXStrepFlua<br>
+
9. pMALongLFoki<br />
-
<br>*Preparative Gel of Digest<br>
+
10.pEXStrepFlua<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]]
+
</div>
-
Freiburg09_041009_umklonierung_gel.JPG
+
<br />
-
<br>*Gelextraction<br>
+
<ul>
-
<br>*Ligation of<br>
+
  <li>Preparative Gel of Digest</li>
-
1.pMAHisDigMiddleFoki<br>
+
</ul>
-
2.pMACAT<br>
+
<div style="margin-left: 40px;">
-
3.pEXStrepFluAShortLFokA<br>
+
<table style="text-align: left; width: 750px;" border="0"
-
4.pEXStrepFluAShortLFoki<br>
+
cellpadding="0" cellspacing="0">
-
5.pEXStrepFluAmiddleLFokA<br>
+
  <tbody>
-
6.pEXStrepFluAMiddleLFoki<br>
+
    <tr>
-
7.pEXStrepFluALongLFokA<br>
+
      <td><img style="width: 759px; height: 575px;"
-
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>Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki,
-
<br>*Dephosphorylation of pBAD Vector: <br>
+
4.pMASplitFoki,
-
Approach: 2µl Eluat, 3,1 µl Fast Ap Buffer 10x, 1µl Fast Ap, 10 min. 37°C, 5 min. 75°C<br>
+
5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli,
-
<br>*Transformation of<br>
+
9.pMALongli, 10.pExStrepFluA</td>
-
1.pMAHisDigMiddleFoki (RV)<br>
+
    </tr>
-
2.pMACAT(RV)<br>
+
  </tbody>
-
3.pEXStrepFluAShortLFokA(RV)<br>
+
</table>
-
4.pEXStrepFluAShortLFoki(RV)<br>
+
<br />
-
5.pEXStrepFluAmiddleLFokA(RV)<br>
+
</div>
-
6.pEXStrepFluAMiddleLFoki(RV)<br>
+
<br />
-
7.pEXStrepFluALongLFokA(RV)<br>
+
<ul>
-
8.pEXStrepFluALongLFoki(RV)<br>
+
  <li>Gelextraction</li>
-
9.pBAD (RV)<br>
+
  <li>Ligation of</li>
-
10.pJS 419StrepDigSplitFokA(XBL)<br>
+
</ul>
-
11.pJS 419HisDigSplitFokA(XBL)<br>
+
<div style="margin-left: 40px;">
-
12.pJS 418HisDigSplitFokA(XBL)<br>
+
1.pMAHisDigMiddleFoki<br />
-
<br>*test digest of plasmid preps from today
+
2.pMACAT<br />
-
-> run on a gel with digest pET39, xbaI, ecoRI from Tobi
+
3.pEXStrepFluAShortLFokA<br />
-
<br>*starter culture of pEx_DsbA_HisDigSplitFoka
+
4.pEXStrepFluAShortLFoki<br />
-
-> modified expression has to be done tomorrow
+
5.pEXStrepFluAmiddleLFokA<br />
-
<br>*inoculation of pJS418 and pJS419 for glycerolstocks tomorrow
+
6.pEXStrepFluAMiddleLFoki<br />
-
 
+
7.pEXStrepFluALongLFokA<br />
-
<br>*Taq AGO BB PCR did not work
+
8.pEXStrepFluALongLFoki<br />
-
 
+
9.JS 419StrepDigSplitFokA<br />
-
<br>*Phageproduction day 2
+
10.JS 419HisDigSplitFokA<br />
-
 
+
11.JS 418HisDigSplitFokA<br />
-
<h3>05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika</h3>
+
Approach: 8&micro;l H2O, 3 &micro;l Vector, 6&micro;l
-
 
+
Insert, 2&micro;l Quick Ligase Buffer, 1 &micro;l Quick Ligase,
-
<br>* 3l LB and 2L LB Agar
+
15 min, Room Temp.<br />
-
 
+
</div>
-
<br>*Periplasma Project
+
<br />
-
 
+
<ul>
-
<br>*digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09)
+
  <li>Dephosphorylation of pBAD Vector: </li>
-
<br>*test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka
+
</ul>
-
<br>*1) Gelextraction<br>
+
<div style="margin-left: 40px;">Approach: 2&micro;l
-
 
+
Eluat, 3,1 &micro;l Fast Ap Buffer 10x, 1&micro;l Fast Ap, 10
-
--> Gelbild <br>
+
min. 37&deg;C, 5 min. 75&deg;C<br />
-
 
+
</div>
-
1) Vector: pEXHisFluSplitFoki<br>
+
<br />
-
2)Insert: RBSStrepDigSplitFokA<br>
+
<ul>
-
3)PET39b+<br>
+
  <li>Transformation of</li>
-
 
+
</ul>
-
<br>*2)Ligation<br>
+
<div style="margin-left: 40px;">1.pMAHisDigMiddleFoki (RV)<br />
-
-10ul 2 fold buffer<br>
+
2.pMACAT(RV)<br />
-
- 6 µl Insert<br>
+
3.pEXStrepFluAShortLFokA(RV)<br />
-
-3µl Vector<br>
+
4.pEXStrepFluAShortLFoki(RV)<br />
-
-1µl Quick Ligase<br>
+
5.pEXStrepFluAmiddleLFokA(RV)<br />
-
 
+
6.pEXStrepFluAMiddleLFoki(RV)<br />
-
--> 15 min at RT<br>
+
7.pEXStrepFluALongLFokA(RV)<br />
-
 
+
8.pEXStrepFluALongLFoki(RV)<br />
-
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA<br>
+
9.pBAD (RV)<br />
-
 
+
10.pJS 419StrepDigSplitFokA(XBL)<br />
-
<br>*3)Transformation<br>
+
11.pJS 419HisDigSplitFokA(XBL)<br />
-
-5 µl DNA of Ligation + BL21d3 and XBL
+
12.pJS 418HisDigSplitFokA(XBL)<br />
-
 
+
</div>
-
<br>*'''Mini prep with with Qiagen Spin Miniprep Kit of'''
+
<br />
-
 
+
<ul>
-
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3<br>
+
  <li>test digest of plasmid preps from today
-
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br>
+
-&gt; run on a gel with digest pET39, xbaI, ecoRI from Tobi
-
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br>
+
  </li>
-
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br>
+
  <li>starter culture of pEx_DsbA_HisDigSplitFoka
-
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br>
+
-&gt; modified expression has to be done tomorrow
-
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br>
+
  </li>
-
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br>
+
  <li>inoculation of pJS418 and pJS419 for glycerolstocks
-
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br>
+
tomorrow
-
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br>
+
  </li>
-
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br>
+
  <li>Taq AGO BB PCR did not work
-
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br>
+
  </li>
-
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br>
+
  <li>Phageproduction day 2
 +
  </li>
 +
</ul>
 +
<h3>05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes,
 +
Gerrit, Anika</h3>
 +
<br />
 +
<ul>
 +
  <li>&nbsp;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;">--&gt; Gelbild <br />
 +
1) Vector: pEXHisFluSplitFoki<br />
 +
2)Insert: RBSStrepDigSplitFokA<br />
 +
3)PET39b+<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>2)Ligation</li>
 +
</ul>
 +
<div style="margin-left: 40px;">-10ul 2 fold buffer<br />
 +
- 6 &micro;l Insert<br />
 +
-3&micro;l Vector<br />
 +
-1&micro;l Quick Ligase<br />
 +
--&gt; 15 min at RT<br />
 +
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>3)Transformation</li>
 +
</ul>
 +
<div style="margin-left: 40px;">-5 &micro;l DNA of
 +
Ligation + BL21d3 and XBL
 +
<br />
 +
</div>
 +
<ul>
 +
  <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;">-
 +
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
-
 
+
<br />
-
<br>*'''Made Glycerolstocks of'''
+
</div>
-
 
+
<ul>
-
<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>700&micro;l Cellsuspension+ 300&micro;l 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;">-
-
- 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
-
 
+
<br />
-
<br>*'''Test Digestion of'''
+
</div>
-
 
+
<ul>
-
<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: 5&micro;l DNA, 2,5&micro;l H2O,
-
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1<br>
+
1&micro;l Buffer 3, 1&micro;l EcoRI, 1&micro;l PstI,
-
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1<br>
+
1&micro;l 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;">-
-
- 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<br />
-
- induced with 0,7mM IPTG and took samples T0-T5<br>
+
</div>
-
- centrifuged in buckets 17', 4000rpm, 4°C<br>  
+
<br />
-
- eluted in 20 ml TES in each bucket<br>
+
<ul>
 +
  <li>&nbsp;Transformation of</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- XBL
 +
pEXHisFluASplitFokIRBSStrepDigSplitFokA<br />
 +
- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <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;">- centrifuged in buckets
 +
17', 4000rpm, 4&deg;C<br />
 +
- eluted in 20 ml TES in each bucket<br />
....
....
-
 
+
<br />
-
<br>*PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -> very few product, as well as non specific ones
+
</div>
-
<br>*PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature
+
<ul>
-
 
+
  <li>PET 39 b+ ssDNA "PCR" with new template from digestion of
-
<h3>06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika</h3>
+
yesterday -&gt; very few product, as well as non specific ones
-
<br>*digest
+
  </li>
-
- pBAD vector <br>
+
  <li>PET 39 b+ ssDNA "PCR" with more cycles and higher annealing
-
DNA: 16.8µl<br>
+
temperature
-
enzyme: 1µl AgeI<br>
+
  </li>
-
buffer: 2µl buffer 1 <br>
+
</ul>
-
 
+
<h3>06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes,
-
- pBAD insert <br>
+
Gerrit, Anika</h3>
-
DNA: 16.8µl<br>
+
<br />
-
enzyme: 1µl XmaI<br>
+
<ul>
-
buffer: 2µl buffer 4 <br>
+
  <li>digest
-
BSA (10X): 2µl <br>
+
- pBAD vector </li>
-
 
+
</ul>
-
- pJS 418/419 <br>
+
<div style="margin-left: 40px;">DNA: 16.8&micro;l<br />
-
DNA: 10µl<br>
+
enzyme: 1&micro;l AgeI<br />
-
enzyme: 1µl PstI and 1.5 µl Xba/<br>
+
buffer: 2&micro;l buffer 1 <br />
-
buffer: 3µl buffer 3 <br>
+
- pBAD insert <br />
-
BSA (10X): 3µl <br>
+
DNA: 16.8&micro;l<br />
-
 
+
enzyme: 1&micro;l XmaI<br />
-
- pExStrepDigSplitFoka/pExHisDigSplitFoka <br>
+
buffer: 2&micro;l buffer 4 <br />
-
DNA: 16.8µl<br>
+
BSA (10X): 2&micro;l <br />
-
enzyme: 1µl PstI and 1.5 µl Xba/<br>
+
- pJS 418/419 <br />
-
buffer: 3µl buffer 3 <br>
+
DNA: 10&micro;l<br />
-
BSA (10X): 3µl <br>
+
enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/<br />
-
 
+
buffer: 3&micro;l buffer 3 <br />
-
<br>*testdigest of pExHisFluASplitFoki_StrepDigSplitFoka  
+
BSA (10X): 3&micro;l <br />
-
DNA: 5µl<br>
+
- pExStrepDigSplitFoka/pExHisDigSplitFoka <br />
-
enzyme: 1µl PstI and 1.5 µl Xba/<br>
+
DNA: 16.8&micro;l<br />
-
buffer: 1µl buffer 3 <br>
+
enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/<br />
-
BSA (10X): 1µl <br>
+
buffer: 3&micro;l buffer 3 <br />
-
<br>*preparative gels of digests
+
BSA (10X): 3&micro;l <br />
-
<table border="0">
+
</div>
-
<tr><td>
+
<br />
-
[[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]]
+
<ul>
-
</td>
+
  <li>testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA:
-
<td>
+
5&micro;l</li>
-
[[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]]
+
</ul>
-
</td></tr></table>
+
<div style="margin-left: 40px;">enzyme: 1&micro;l PstI
-
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
+
and 1.5 &micro;l Xba/<br />
-
<br>*testdigest of pMAdsba
+
buffer: 1&micro;l buffer 3 <br />
-
DNA: 5µl<br>
+
BSA (10X): 1&micro;l <br />
-
enzyme: 1µl PstI and 1.5 µl Xba/<br>
+
</div>
-
buffer: 1µl buffer 3 <br>
+
<br />
-
BSA (10X): 1µl <br>
+
<br />
-
 
+
<ul>
-
<br>*His-tag purification of HisDigSplitFoka (periplasm export) with Ni-NTA column. Washing buffer: 25mM imidazole
+
  <li>preparative gels of digests
-
-cells were sonicated for 2 x 1min before filtering with 0.45µm and 0.22µm filter
+
  </li>
-
 
+
</ul>
-
<br>*Ligation
+
<div style="margin-left: 80px;"></div>
-
- Dephphorylation of pBAD Gelex<br>
+
<table style="text-align: left; width: 750px; margin-left: 40px;"
-
- 1µl Fast AP<br>
+
border="1" cellpadding="0" cellspacing="1">
-
- 5.5µl fast AP buffer<br>
+
  <tbody>
-
-1.5µl water<br>
+
    <tr>
-
 
+
      <td><img class="art-article"
-
--> Solution was given to eluat
+
style="width: 350px; height: 280px;" alt=""
-
 
+
src="https://static.igem.org/mediawiki/2009/d/d5/Freiburg09_061009_bad_js.JPG" /></td>
-
- for each ligation:<br>
+
      <td><img class="art-article"
-
- 6µl Insert<br>
+
style="width: 423px; height: 280px;" alt=""
-
-3µl Vetor<br>
+
src="https://static.igem.org/mediawiki/2009/7/75/Freiburg09_061009_js_foka.JPG" /></td>
-
-1µl Quickligase<br>
+
    </tr>
-
-10µl buffer<br>
+
    <tr>
-
 
+
      <td>preparative agarose gel, lanes: 1. Genruler DNA ladder
-
--> pBAD <br>* Insert (Dummy)<br>
+
mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418</td>
-
--> pJS419+HisDigSplitFoka
+
      <td>preparative agarose gel, lanes: 1. Genruler DNA ladder
-
-->pJ418+HisDigSplitFoka
+
mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka</td>
-
<br>* Transformation
+
    </tr>
-
 
+
  </tbody>
-
--> pBAD <br>* Insert (Dummy)<br>
+
</table>
-
--> pJS419+HisDigSplitFoka
+
<div style="margin-left: 40px;">interpretation:
-
-->pJ418+HisDigSplitFoka
+
pExStrepDigSplitFoka seemed
-
 
+
to be a wrong construct, the other constructs showed bands of the right
-
- in XLblue<br>
+
size even if the concentration of the PCR pBAD insert seems to be very
-
 
+
low
-
-pBAD was plated on AMP plates<br>
+
<br />
-
- Both pJS... were plated on CM plates<br>
+
</div>
-
 
+
<ul>
-
<br>*'''Ligation and Transformation of'''
+
  <li>testdigest of pMAdsba
-
 
+
DNA: 5&micro;l</li>
-
 
+
</ul>
-
Ligation:per sample 8µl H2O; 3µl vector; 6µl insert; 2µl Quick Ligase buffer; 1µl Quick Ligase
+
<div style="margin-left: 40px;">enzyme: 1&micro;l PstI
-
 
+
and 1.5 &micro;l Xba/<br />
-
 
+
buffer: 1&micro;l buffer 3 <br />
-
Transformation:<b>Defrost competent cells on ice:</b>(100 µl);
+
BSA (10X): 1&micro;l <br />
-
<b>add of the ligation:</b> 5 µl;
+
<br />
-
<b>DNA and cells:</b> mix softly by knocking;  
+
</div>
-
<b>Incubation on ice for:</b> 20-30 min;
+
<ul>
-
<b>Heat shock at:</b> 42°C for 40 sec;
+
  <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
 +
0.45&micro;m and 0.22&micro;m filter
 +
  </li>
 +
</ul>
 +
<ul>
 +
  <li>Ligation
 +
- Dephphorylation of pBAD Gelex</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- 1&micro;l Fast AP<br />
 +
- 5.5&micro;l fast AP buffer<br />
 +
-1.5&micro;l water<br />
 +
--&gt; Solution was given to eluat
 +
- for each ligation:<br />
 +
- 6&micro;l Insert<br />
 +
-3&micro;l Vetor<br />
 +
-1&micro;l Quickligase<br />
 +
-10&micro;l buffer<br />
 +
--&gt; pBAD <br />
 +
</div>
 +
<ul>
 +
  <li>&nbsp;Insert (Dummy)</li>
 +
</ul>
 +
<div style="margin-left: 40px;">--&gt;
 +
pJS419+HisDigSplitFoka
 +
--&gt;pJ418+HisDigSplitFoka
 +
<br />
 +
</div>
 +
<ul>
 +
  <li>&nbsp;Transformation
 +
--&gt; pBAD </li>
 +
  <li>&nbsp;Insert (Dummy)</li>
 +
</ul>
 +
<div style="margin-left: 40px;">--&gt;
 +
pJS419+HisDigSplitFoka
 +
--&gt;pJ418+HisDigSplitFoka
 +
- in XLblue<br />
 +
-pBAD was plated on AMP plates<br />
 +
- Both pJS... were plated on CM plates<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li><big>Ligation and Transformation of</big></li>
 +
</ul>
 +
<div style="margin-left: 40px;">&nbsp;Ligation:per
 +
sample 8&micro;l H2O; 3&micro;l vector;
 +
6&micro;l insert; 2&micro;l Quick Ligase buffer; 1&micro;l
 +
Quick Ligase
 +
Transformation:<b>Defrost competent cells on ice:</b>(100
 +
&micro;l);
 +
<b>add of the ligation:</b> 5 &micro;l;
 +
<b>DNA and cells:</b> mix softly by knocking; <b>Incubation
 +
on ice for:</b> 20-30 min;
 +
<b>Heat shock at:</b> 42&deg;C for 40 sec;
<b>Cool off on ice for:</b> 5 min;
<b>Cool off on ice for:</b> 5 min;
-
<b>add sterile LB(or dyt)medium:</b> 900 µl;  
+
<b>add sterile LB(or dyt)medium:</b> 900 &micro;l; <b>Incubation
-
<b>Incubation in(shaker)at:</b> 37°C for 60-70 min;  
+
in(shaker)at:</b> 37&deg;C for 60-70 min; <b>Plate
-
<b>Plate cells on LB+antibiotic plates:</b> ampicillin;
+
cells on LB+antibiotic plates:</b> ampicillin;
-
<b>ligation:</b> 2 plates: 1. 50µl cells  
+
<b>ligation:</b> 2 plates: 1. 50&micro;l cells <b>2.
-
<b>2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the restcells and plate out:</b>  
+
centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the
-
 
+
restcells and plate out:</b> -pMA HisDig Middle Linker Foki<br />
-
-pMA HisDig Middle Linker Foki<br>
+
<br />
-
-pMA CAT<br>
+
</div>
-
-pEX Strep FluA SL FokA<br>
+
<div style="margin-left: 40px;">-pMA CAT<br />
-
-pEX Strep FluA LL Foki<br>
+
-pEX Strep FluA SL FokA<br />
-
-pEX Strep FluA ML Foki<br>
+
-pEX Strep FluA LL Foki<br />
-
-pEX Strep FluA ML Foka<br>
+
-pEX Strep FluA ML Foki<br />
-
-pEX Strep FluA LL Foka<br>
+
-pEX Strep FluA ML Foka<br />
-
 
+
</div>
-
 
+
<div style="margin-left: 40px;">-pEX Strep FluA LL Foka<br />
-
<br>*Sequencing:
+
</div>
-
 
+
<br />
-
27µl H2O; 3µl DNA
+
<ul>
-
 
+
  <li>Sequencing:
-
-pMA Dbsa clone 2<br>
+
27&micro;l H2O; 3&micro;l DNA
-
-pMA HisDigSplitFokA clone 2<br>
+
-pMA Dbsa clone 2</li>
-
-pMA HisFluASL Foki clone 1<br>
+
</ul>
-
-pMA HisFluASplitFoki clone 2<br>
+
<div style="margin-left: 40px;">-pMA HisDigSplitFokA clone
-
-pMA StrepDigLLFoki clone 3<br>
+
2<br />
-
-pMA HisFluaMLFoki clone 3<br>
+
-pMA HisFluASL Foki clone 1<br />
-
-pMA StrepDigSplitFokA clone 1<br>
+
-pMA HisFluASplitFoki clone 2<br />
-
-pMA StrepDigMLFoki clone 1<br>
+
-pMA StrepDigLLFoki clone 3<br />
-
-pMA StrepDigSLFoki clone 2<br>
+
-pMA HisFluaMLFoki clone 3<br />
-
-pMA HisDigLLFoki clone 1<br>
+
-pMA StrepDigSplitFokA clone 1<br />
-
-pMA StrepDigSplitFoki clone 3<br>
+
-pMA StrepDigMLFoki clone 1<br />
-
-pMA HisDigSplitFoki clone 1<br>
+
-pMA StrepDigSLFoki clone 2<br />
-
-pMA HisFluaLLFoki clone 3<br>
+
-pMA HisDigLLFoki clone 1<br />
-
 
+
-pMA StrepDigSplitFoki clone 3<br />
-
<br>*Inoculation
+
-pMA HisDigSplitFoki clone 1<br />
-
 
+
-pMA HisFluaLLFoki clone 3<br />
-
of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room overnight<br>
+
</div>
-
 
+
<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)
+
<ul>
-
 
+
  <li>Inoculation
-
<br>*New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1 and A4 _see gele_
+
of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37&deg;c room
-
no differend results were obtained compaired to the assays using unphorphorylated oligos
+
overnight</li>
-
 
+
  <li>Yesterdays improved pcr resulted in a lot of product, but
-
<br>*Started dialysis to transfer the leftover AGO-proteins into the assay buffer
+
still
-
 
+
unspecific ones.. made new one with even higher annealing temperature
 +
(69&deg;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>
<h3>07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika</h3>
<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
+
<br />
-
 
+
<ul>
-
 
+
  <li>SDS gel of protein purification of HisDigSplitFoka
-
[[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]]
+
(periplasm)
-
 
+
from 06.10.09
-
 
+
[[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS
-
<br>* Inoculation
+
gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker
-
4 Clones respectively:<br>
+
broad range, Elution fraction 1, Elution fraction 2, Elution fraction
-
- pMA-His-Dig-MiddleLinker-Foki in XLBlue<br>
+
3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution
-
- pJS419-HIs-Dig-Split-Foka- in XLBlue<br>
+
fraction 6, flow through fraction 3, washing fraction 2, periplasm
-
- pJS418-HIs-DIg-Split-Foka- in XLBlue<br>
+
extract (frozen over night at -80&deg;C)|400x400px]]
-
- pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest<br>
+
  </li>
-
- pEX-Strep-FluA-LongLinker-Foki in XL1blue rest<br>
+
</ul>
-
- pMA-CAT in XLblue 10µl<br>
+
<ul>
-
-pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest<br>
+
  <li>&nbsp;Inoculation 4 Clones respectively:</li>
-
 
+
</ul>
-
2 Clones respectively:<br>
+
<div style="margin-left: 40px;">-
 +
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 10&micro;l<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
-
 
+
<br />
-
 
+
</div>
-
<br>* Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3
+
<ul>
-
<br>*two step PCR assembly of DsbA, His_Fos, and SplitFoka
+
  <li>&nbsp;Starter culture of pEx-DsbAHisDigSplitFoka in
-
- program name: Assembly<br>
+
Bl21de3
-
- three different samples: 1. without DMSO, 2. with DMSO, 3. without DMSO and with last primers just added after first step
+
  </li>
-
<br>*preparative gel of the PCR samples
+
  <li>two step PCR assembly of DsbA, His_Fos, and SplitFoka
-
-> primer haven't been diluted an probably made all secondary structures, has to be repeated
+
- program name: Assembly</li>
-
<br>*digest of pBAD with AgeI and new PCR with insert digested with XmaI
+
</ul>
-
<br>*preparative gel with digest and  
+
<div style="margin-left: 40px;">- three different samples:
-
<br>*test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka  
+
1. without DMSO, 2. with DMSO, 3. without
-
DNA: 5µl<br>
+
DMSO and with last primers just added after first step
-
Enzymes: 0.5µl of BamHI and MfeI each<br>
+
<br />
-
Buffer: 1µl buffer 4<br>
+
</div>
-
BSA (10fold): 1µl<br>
+
<ul>
-
Water: 2µl<br>
+
  <li>preparative gel of the PCR samples
-
-> pJS419_HisDigSplitFoka showed bands of the right size and was sequenced with primer sf_lac P1<br>
+
-&gt; primer haven't been diluted an probably made all secondary
-
<br>*plasmidpreparation of  
+
structures, has to be repeated
-
- pEXHisFluASplitFoki<br>
+
  </li>
-
- pExStrepDigSplitFoka<br>
+
  <li>digest of pBAD with AgeI and new PCR with insert digested
-
-> low concentrations<br>
+
with XmaI
-
<br>*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka
+
  </li>
-
pExHisFluASplitFoki: 15µl<br>
+
  <li>preparative gel with digest and </li>
-
Enzymes: 1µl of PstI and 1.5µl SpeI<br>
+
  <li>test digest of pJS419_HisDigSplitFoka and
-
Buffer: 3µl buffer 2<br>
+
pJS419_StrepDigSplitFoka
-
BSA (100fold): 0.5µl<br>
+
DNA: 5&micro;l</li>
-
Water: 9µl<br>
+
</ul>
-
 
+
<div style="margin-left: 40px;">Enzymes: 0.5&micro;l
-
pExStrepDigSplitFoka: 15µl<br>
+
of BamHI and MfeI each<br />
-
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
+
Buffer: 1&micro;l buffer 4<br />
-
Buffer: 3µl buffer 3<br>
+
BSA (10fold): 1&micro;l<br />
-
BSA (100fold): 0.5µl<br>
+
Water: 2&micro;l<br />
-
Water: 9µl<br>
+
-&gt; pJS419_HisDigSplitFoka showed bands of the right size and was
-
 
+
sequenced with primer sf_lac P1<br />
-
pExStrepDigSplitFoka: 10µl<br>
+
</div>
-
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
+
<br />
-
Buffer: 3µl buffer 3<br>
+
<ul>
-
BSA (100fold): 0.5µl<br>
+
  <li>plasmidpreparation of - pEXHisFluASplitFoki</li>
-
Water: 14µl<br>
+
</ul>
-
<br>* Gelextraction of digest
+
<div style="margin-left: 40px;">- pExStrepDigSplitFoka<br />
-
1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) <br>
+
-&gt; low concentrations<br />
-
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br>  
+
</div>
-
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br>
+
<br />
-
 
+
<ul>
-
 
+
  <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and
-
<br>*preparative gels of digests
+
RBS_StrepDigSplitFoka
-
<table border="0">
+
pExHisFluASplitFoki: 15&micro;l</li>
-
<tr><td>
+
</ul>
-
[[Image:|none|thumb|preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product]]
+
<div style="margin-left: 40px;">Enzymes: 1&micro;l of
-
</td>
+
PstI and 1.5&micro;l SpeI<br />
-
<td>
+
Buffer: 3&micro;l buffer 2<br />
-
[[Image: |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
+
BSA (100fold): 0.5&micro;l<br />
-
</td></tr></table>  
+
Water: 9&micro;l<br />
-
 
+
pExStrepDigSplitFoka: 15&micro;l<br />
-
RBS PCR Product: m= 90 mg<br>
+
Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI<br />
-
vector: 0 0 130 mg<br>
+
Buffer: 3&micro;l buffer 3<br />
-
 
+
BSA (100fold): 0.5&micro;l<br />
-
<br>* Ligation
+
Water: 9&micro;l<br />
-
- 6µl Insert<br>
+
pExStrepDigSplitFoka: 10&micro;l<br />
-
-3µl Vetor<br>
+
Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI<br />
-
-1µl Quickligase<br>
+
Buffer: 3&micro;l buffer 3<br />
-
-10µl buffer<br>
+
BSA (100fold): 0.5&micro;l<br />
-
 
+
Water: 14&micro;l<br />
-
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br>
+
</div>
-
<br>*Transformation
+
<br />
-
 
+
<ul>
-
- All in XL1blue<br>
+
  <li>&nbsp;Gelextraction of digest
-
- Vector: pEX-strep-Duig-Split-Foka<br>
+
1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) </li>
-
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br>
+
</ul>
-
 
+
<div style="margin-left: 40px;">2)Vector:
-
<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
+
pEX-His-Flua-Split-Foki (digested with spe, pst)<br />
-
<br>*prepaired ELISA with anti-flag antibodies
+
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br />
-
<br>*made electro competent cells for transformation with the ago phagmidbibliothek
+
</div>
-
 
+
<br />
 +
<ul>
 +
  <li>preparative gels of digests
 +
  </li>
 +
</ul>
 +
<table style="margin-left: 40px;" border="0">
 +
  <tbody>
 +
    <tr>
 +
      <td>[[Image:|none|thumb|preparative agarose gel, lanes: 1.
 +
Insert2. pEX-Vector, 3. PCR- RBS Product]]
 +
      </td>
 +
      <td>[[Image: |none|thumb|preparative agarose gel, lanes: 1.
 +
Genruler
 +
DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4.
 +
pExStrepDigSplitFoka |300x300px]]
 +
      </td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<div style="margin-left: 40px;">RBS PCR Product: m= 90 mg<br />
 +
vector: 0 0 130 mg<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>&nbsp;Ligation
 +
- 6&micro;l Insert</li>
 +
</ul>
 +
<div style="margin-left: 40px;">-3&micro;l Vetor<br />
 +
-1&micro;l Quickligase<br />
 +
-10&micro;l buffer<br />
 +
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>Transformation
 +
- All in XL1blue</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- Vector:
 +
pEX-strep-Duig-Split-Foka<br />
 +
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>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>
 +
  <li>prepaired ELISA with anti-flag antibodies
 +
  </li>
 +
  <li>made electro competent cells for transformation with the
 +
ago
 +
phagmidbibliothek
 +
  </li>
 +
</ul>
<h3>08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes </h3>
<h3>08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes </h3>
-
<br>*Poured SDS-Gels<br>
+
<br />
-
<br>*Phage ss DNA  
+
<ul>
-
 
+
  <li>Poured SDS-Gels</li>
-
-Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet<br>
+
  <li>Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet
-
-Transfomation with 3µl M13 Phage stock (-80°) at 10:30Uhr , incubated for 4-5 hours at 37°C in shaker<br>
+
dilute on
-
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C<br>
+
OD600=0,4 in 50ml LB+Tet</li>
-
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml precipitation over night on ice in -4°C room<br>
+
</ul>
-
 
+
<div style="margin-left: 40px;">
-
<br>*Miniprep
+
-Transfomation with 3&micro;l M13 Phage stock (-80&deg;) at
-
 
+
10:30Uhr , incubated for 4-5 hours at 37&deg;C in shaker<br />
-
- 2 clones respectively:<br>
+
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4&deg;C<br />
-
-A = pEX strep dig split foka
+
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml
-
 
+
precipitation over night on ice in -4&deg;C room<br />
-
<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>
+
</div>
-
 
+
<br />
-
<br>*Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):<br>
+
<ul>
-
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.
+
  <li>Miniprep
-
 
+
- 2 clones respectively:</li>
-
<>     1     2     3     4     5     6     7     8     9     10   11   12    <br>
+
</ul>
-
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>
+
<div style="margin-left: 40px;">-A = pEX strep dig split
-
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>
+
foka
-
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>
+
<br />
-
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>
+
</div>
-
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>
+
<ul>
-
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>
+
  <li>Finished Phage production(see protocol day 2): We obtained
-
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>
+
approximately 3.8</li>
-
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>
+
  <li>10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1</li>
-
 
+
  <li>10^13 pJS_TorA-flag-AGO-CDg3p (448) -&gt; aplyed all of
-
<br>* Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction
+
them to
-
 
+
the anti-Flag ELISA</li>
-
<br>*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka
+
</ul>
-
pExHisFluASplitFoki: 15µl<br>
+
<br />
-
Enzymes: 1µl of PstI and 1.5µl SpeI<br>
+
<ul>
-
Buffer: 3µl buffer 2<br>
+
  <li>Anti-Flag ELISA was successfull with a slight but detctable
-
BSA (100fold): 0.5µl<br>
+
signal via
-
Water: 9µl<br>
+
anti M13 VCS Antibodies (with peroxidase):</li>
-
 
+
</ul>
-
pExHisDigSplitFoka: 15µl<br>
+
<div style="margin-left: 40px;">see
-
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
+
405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to
-
Buffer: 3µl buffer 3<br>
+
positive control (G5-6) and negative control (G3-4 and D3-10) detected
-
BSA (100fold): 0.5µl<br>
+
about half an hour after the ABTS substrat was addet.
-
Water: 14µl<br>
+
<br />
-
 
+
<br />
-
RBSStrepDigSplitFoka: 5µl<br>
+
<table id="table_1" border="2" cellpadding="2"
-
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
+
cellspacing="0">
-
Buffer: 3µl buffer 3<br>
+
  <tbody>
-
BSA (100fold): 0.5µl<br>
+
    <tr>
-
Water: 19µl<br>
+
      <td>&nbsp;</td>
-
->made two digest of PCR construct<br>
+
      <td>1</td>
-
<br>* Gelextraction of digest
+
      <td>2</td>
-
1) Insert: His-Dig-Split-Foka( digested with xba and pst) <br>
+
      <td>3</td>
-
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br>  
+
      <td>4</td>
-
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br>
+
      <td>5</td>
 +
      <td>6</td>
 +
      <td>7</td>
 +
      <td>8</td>
 +
      <td>9</td>
 +
      <td>10</td>
 +
      <td>11</td>
 +
      <td>12</td>
 +
     </tr>
 +
    <tr>
 +
      <td>A</td>
 +
      <td>0.0470</td>
 +
      <td>0.0420</td>
 +
      <td>0.0440</td>
 +
      <td>0.0430</td>
 +
      <td>0.0480</td>
 +
      <td>0.0510</td>
 +
      <td>0.0460</td>
 +
      <td>0.0470</td>
 +
      <td>0.0470</td>
 +
      <td>0.0480</td>
 +
      <td>0.0450</td>
 +
      <td>0.0460</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B</td>
 +
      <td>0.0470</td>
 +
      <td>0.0460</td>
 +
      <td>0.0480</td>
 +
      <td>0.0480</td>
 +
      <td>0.0460</td>
 +
      <td>0.0450</td>
 +
      <td>0.0470</td>
 +
      <td>0.0560</td>
 +
      <td>0.0470</td>
 +
      <td>0.0450</td>
 +
      <td>0.0450</td>
 +
      <td>0.0440</td>
 +
    </tr>
 +
    <tr>
 +
      <td>C</td>
 +
      <td>0.0460</td>
 +
      <td>0.0470</td>
 +
      <td>0.0500</td>
 +
      <td>0.0450</td>
 +
      <td>0.0430</td>
 +
      <td>0.0460</td>
 +
      <td>0.0450</td>
 +
      <td>0.0460</td>
 +
      <td>0.0430</td>
 +
      <td>0.0420</td>
 +
      <td>0.0440</td>
 +
      <td>0.0470</td>
 +
    </tr>
 +
    <tr>
 +
      <td>D</td>
 +
      <td>0.0490</td>
 +
      <td>0.0470</td>
 +
      <td>0.1590</td>
 +
      <td>0.1610</td>
 +
      <td>0.1920</td>
 +
      <td>0.1720</td>
 +
      <td>0.2210</td>
 +
      <td>0.2500</td>
 +
      <td>0.2130</td>
 +
      <td>0.2680</td>
 +
      <td>0.0460</td>
 +
      <td>0.0480</td>
 +
    </tr>
 +
    <tr>
 +
      <td>E</td>
 +
      <td>0.0490</td>
 +
      <td>0.0490</td>
 +
      <td>0.0450</td>
 +
      <td>0.0490</td>
 +
      <td>0.0430</td>
 +
      <td>0.0490</td>
 +
      <td>0.0460</td>
 +
      <td>0.0430</td>
 +
      <td>0.0460</td>
 +
      <td>0.0420</td>
 +
      <td>0.0450</td>
 +
      <td>0.0440</td>
 +
    </tr>
 +
    <tr>
 +
      <td>F</td>
 +
      <td>0.0490</td>
 +
      <td>0.0430</td>
 +
      <td>0.0470</td>
 +
      <td>0.0440</td>
 +
      <td>0.0470</td>
 +
      <td>0.0470</td>
 +
      <td>0.0440</td>
 +
      <td>0.0450</td>
 +
      <td>0.0500</td>
 +
      <td>0.0460</td>
 +
      <td>0.0450</td>
 +
      <td>0.0450</td>
 +
    </tr>
 +
    <tr>
 +
      <td>G</td>
 +
      <td>0.0500</td>
 +
      <td>0.0440</td>
 +
      <td>0.2530</td>
 +
      <td>0.2510</td>
 +
      <td>3.9180</td>
 +
      <td>3.8800</td>
 +
      <td>0.9560</td>
 +
      <td>0.9380</td>
 +
      <td>1.2420</td>
 +
      <td>1.2360</td>
 +
      <td>0.0440</td>
 +
      <td>0.0460</td>
 +
    </tr>
 +
    <tr>
 +
      <td>H</td>
 +
      <td>0.0480</td>
 +
      <td>0.0430</td>
 +
      <td>0.0510</td>
 +
      <td>0.0470</td>
 +
      <td>0.0480</td>
 +
      <td>0.0460</td>
 +
      <td>0.0470</td>
 +
      <td>0.0450</td>
 +
      <td>0.0450</td>
 +
      <td>0.0440</td>
 +
      <td>0.0460</td>
 +
      <td>0.0460</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
&nbsp; &nbsp;&nbsp;&nbsp;<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>&nbsp;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: 15&micro;l</li>
 +
</ul>
 +
<div style="margin-left: 40px;">Enzymes: 1&micro;l of
 +
PstI and 1.5&micro;l SpeI<br />
 +
Buffer: 3&micro;l buffer 2<br />
 +
BSA (100fold): 0.5&micro;l<br />
 +
Water: 9&micro;l<br />
 +
pExHisDigSplitFoka: 15&micro;l<br />
 +
Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI<br />
 +
Buffer: 3&micro;l buffer 3<br />
 +
BSA (100fold): 0.5&micro;l<br />
 +
Water: 14&micro;l<br />
 +
RBSStrepDigSplitFoka: 5&micro;l<br />
 +
Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI<br />
 +
Buffer: 3&micro;l buffer 3<br />
 +
BSA (100fold): 0.5&micro;l<br />
 +
Water: 19&micro;l<br />
 +
-&gt;made two digest of PCR construct<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>&nbsp;Gelextraction of digest
 +
1) Insert: His-Dig-Split-Foka( digested with xba and pst) </li>
 +
</ul>
 +
<div style="margin-left: 40px;">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
+
<br />
-
->see picture<br>
+
</div>
-
<br>*ligation of
+
<ul>
-
-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>
+
-&gt;see picture</li>
-
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR purification)<br>
+
</ul>
-
<br>*transformation of ligations
+
<br />
-
<br>*inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol
+
<ul>
-
<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
+
  <li>ligation of
-
->interpretation: no band of 921bp was visible<br>
+
-pEX-His-Flua-Split-Foki_His-Dig-Split-Foka</li>
-
<br>*one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel
+
</ul>
-
->see picture<br>
+
<div style="margin-left: 40px;">-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka
-
->interpretation: pMAFos and pMASplitFoka showed bands of the right size (230bp and 657bp respectively), of pMADsbA no product was visible<br>
+
(from gelex)<br />
-
<br>*new one step PCR of pMADsba with newly prepared 1:1000 dilution
+
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR
-
<br>*test digest of plasmidpreparations from today
+
purification)<br />
-
->see picture<br>
+
</div>
-
 
+
<br />
-
<h3>09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika </h3>
+
<ul>
-
 
+
  <li>transformation of ligations
-
<br>*Phage ss DNA<br>
+
  </li>
-
-Over night in 4°C room precipitated phages: centrifuged for 20 min at 5000rpm and 4°C<br>
+
  <li>inoculation of pJS419_HisDigSplitFoka in LB +
-
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet recognized)<br>
+
chloramphenicol
-
-Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at 13000rpm<br>
+
  </li>
-
-Decant the supernatant into new eppis, precipitate with 170µl PEG/NaCl and leave for 1 hour on ice<br>
+
  <li>new two step PCR with all three plasmids for Fos construct
-
 
+
(pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution
-
<br>*Miniprep
+
and preparative agarose gel
-
- pJS 419-his-dig-split-foka (clon 1+2)<br>
+
-&gt;interpretation: no band of 921bp was visible</li>
-
- Glycerolstock of clon1 and 2<br>
+
</ul>
-
- 300µl Glycerol<br>
+
<br />
-
- 700 µl culture<br>
+
<ul>
-
 
+
  <li>one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and
-
- Pellets from clones 3,5,6<br>
+
preparative agarose gel
-
 
+
-&gt;see picture</li>
-
Nanodrop data:<br>  
+
</ul>
-
pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl<br>
+
<div style="margin-left: 40px;">-&gt;interpretation:
-
pJS 419-his-dig-split-foka-clon2 =468 ng/µl<br>
+
pMAFos and pMASplitFoka showed bands of the
-
 
+
right
-
<br>*Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room
+
size (230bp and 657bp respectively), of pMADsbA no product was visible<br />
-
<br>*send to sequencing:<br>
+
</div>
-
pMA Dig Plasmidprep 22.07.09 Timo1<br>
+
<br />
-
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br>
+
<ul>
-
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br>
+
  <li>new one step PCR of pMADsba with newly prepared 1:1000
-
pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4<br>
+
dilution
-
pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5<br>
+
  </li>
-
pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6<br>
+
  <li>test digest of plasmidpreparations from today
-
pMA Cat plasmidprep clone1 08.10.09 Timo7<br>
+
-&gt;see picture</li>
-
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8<br>
+
</ul>
-
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9<br>
+
<h3>09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes,
-
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12<br>
+
Timo, Max, Anika </h3>
-
 
+
<br />
-
<br>* concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation
+
<ul>
-
<br>* electrical trafo of the 449 ago phagmid bibliothek into XL1
+
  <li>Phage ss DNA</li>
-
<br>* prepaired immutubes with streptavidin for ago phages test panning tomorrow
+
</ul>
-
 
+
<div style="margin-left: 40px;">-Over night in 4&deg;C
-
 
+
room precipitated phages: centrifuged for 20
-
 
+
min at 5000rpm and 4&deg;C<br />
 +
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet
 +
recognized)<br />
 +
-Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at
 +
13000rpm<br />
 +
-Decant the supernatant into new eppis, precipitate with
 +
170&micro;l PEG/NaCl and leave for 1 hour on ice<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>Miniprep
 +
- pJS 419-his-dig-split-foka (clon 1+2)</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- Glycerolstock of clon1
 +
and 2<br />
 +
- 300&micro;l Glycerol<br />
 +
- 700 &micro;l culture<br />
 +
- Pellets from clones 3,5,6<br />
 +
Nanodrop data:<br />
 +
pJS 419-his-dig-split-foka-clon1= 509.5 ng/&micro;l<br />
 +
pJS 419-his-dig-split-foka-clon2 =468 ng/&micro;l<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml
 +
Tet, over
 +
night in 37&deg;C room
 +
  </li>
 +
  <li>send to sequencing:</li>
 +
</ul>
 +
<div style="margin-left: 40px;">pMA Dig Plasmidprep
 +
22.07.09 Timo1<br />
 +
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br />
 +
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br />
 +
pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4<br />
 +
pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5<br />
 +
pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6<br />
 +
pMA Cat plasmidprep clone1 08.10.09 Timo7<br />
 +
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8<br />
 +
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9<br />
 +
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>&nbsp;concentrated pET39b+ ssDNA via sodiumacetat and
 +
ethanol precipitation
 +
  </li>
 +
  <li>&nbsp;electrical trafo of the 449 ago phagmid
 +
bibliothek into XL1
 +
  </li>
 +
  <li>&nbsp;prepaired immutubes with streptavidin for ago
 +
phages test panning
 +
tomorrow
 +
  </li>
 +
</ul>
<h3>10.10.09 Julia, Caro, Laura, Christoph</h3>
<h3>10.10.09 Julia, Caro, Laura, Christoph</h3>
-
 
+
<br />
-
<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>
+
<ul>
-
<br>*Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM
+
  <li>Measured OD600 of over night ER2738 culture abs.1)0.218
-
<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
+
(1:10);2)0.23
-
<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
+
(1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet</li>
-
<br>*Precipitaion over night in 4°C room
+
  <li>Measured OD600 of Christophs culture:abs. 0.37 (1:10),
-
<br>*plasmidpreparation of
+
diluted to
-
- pBADQuick clones 1-6<br>
+
abs.0.07 in 60ml DYT+Tet+CM
-
- pBADT4 clones 1-6<br>
+
  </li>
-
- pMAYFP clone 1-2<br>
+
  <li>Growed Er2738 up to OD600 abs.0.6 8x 250ml in 1L Erlenmeyer
-
- pMACFP clone 1<br>
+
flasks and
-
- pMASplitlinker clones 1-2<br>
+
infected with 15&micro;l M13 phage, shake for 4 hours at
-
- HisTag clones 1-2 (but test digest was negative -> thrown away)<br<
+
37&deg;C
-
<br>*digest for recloning of pMA constructs:
+
  </li>
-
 
+
  <li>Decanted to 16x50ml falcon tubes, centrifuged for 20min at
-
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>
+
5000rpm
-
Enzymes: 1µl of NgoMIVI and 1.5µl PstI<br>
+
and 4&deg;C, decanted supernatant into new 16x50ml falcons with
-
Buffer: 3µl buffer 1<br>
+
each 7ml
-
BSA (10fold): 3µl<br>
+
PEG/NaCl
-
Water: 10,5µl<br>
+
  </li>
-
 
+
  <li>Precipitaion over night in 4&deg;C room
-
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>
+
  </li>
-
Enzymes: 1µl of AgeI and 1.5µl PstI<br>
+
  <li>plasmidpreparation of
-
Buffer: 3µl buffer 1<br>
+
- pBADQuick clones 1-6</li>
-
BSA (10fold): 3µl<br>
+
</ul>
-
Water: 10,5µl<br>
+
<div 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  
+
- pBADT4 clones 1-6<br />
-
<br>*analytical gel of PCRs -> didn't work  
+
- pMAYFP clone 1-2<br />
-
<br>*preparative gels of digests
+
- pMACFP clone 1<br />
-
-> see picture
+
- pMASplitlinker clones 1-2<br />
-
-> no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more
+
- HisTag clones 1-2 (but test digest was negative -&gt; thrown away)<br />
-
<br>*ligation of  
+
&lt;
-
-pMAStrepDigShortLiFoka<br>
+
<br />
-
-pMAStrepDigMiddleLiFoka<br>
+
</div>
-
-pMAStrepDigLongLiFoka<br>
+
<ul>
-
-pExStrepDigShortLiFoka<br>
+
  <li>digest for recloning of pMA constructs:
-
-pExStrepDigMiddleLiFoka<br>
+
pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka
-
-pExStrepDigLongLiFoka<br>
+
clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from
-
-pMAHisDigShortLiFoka<br>
+
10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09,
-
-pMAHisDigMiddleLiFoka<br>
+
pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10&micro;l</li>
-
-pMAHisDigLongLiFoka<br>
+
</ul>
-
-pMACATNd4 (MQI)<br>
+
<div style="margin-left: 40px;">Enzymes: 1&micro;l of
-
-pMACATNd4 (MQII)<br>
+
NgoMIVI and 1.5&micro;l PstI<br />
-
-pMA Kontrolle (M)<br>
+
Buffer: 3&micro;l buffer 1<br />
-
-> in XLBlue<br>
+
BSA (10fold): 3&micro;l<br />
-
- pEx_CATNd4 (EQI)<br>
+
Water: 10,5&micro;l<br />
-
- pEx_CATNd4 (EQII)<br>
+
pMAHisDig clone 2, prep from 24.08.09, pMAStrepDig clone 2, prep from
-
- pEx_Kontrolle(E)<br>
+
25.09., pExStrepDig clone 2, prep from 25.09.09, pMABB057 from
-
->RV308
+
01.10.09: 10&micro;l each<br />
-
Insert: 6µl <br>
+
Enzymes: 1&micro;l of AgeI and 1.5&micro;l PstI<br />
-
vector: 3µl <br>
+
Buffer: 3&micro;l buffer 1<br />
-
Ouick ligase buffer: 10µl <br>
+
BSA (10fold): 3&micro;l<br />
-
Quick ligase: 1µl <br>
+
Water: 10,5&micro;l<br />
-
+
</div>
-
<br>*transformation of ligations in XBL on LB/Amp/1%glucose plates
+
<br />
-
<br>*cotransformation of
+
<ul>
-
<br>*testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture<br>
+
  <li>PCR assembly with pMADsbA, different approaches (Taq
-
[[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]]
+
Polymerase or
-
<br>*cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates
+
Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and
-
<br>*inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09
+
pMAFos together </li>
-
 
+
  <li>analytical gel of PCRs -&gt; didn't work </li>
 +
  <li>preparative gels of digests
 +
-&gt; see picture
 +
-&gt; 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;">-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 />
 +
-&gt; in XLBlue<br />
 +
- pEx_CATNd4 (EQI)<br />
 +
- pEx_CATNd4 (EQII)<br />
 +
- pEx_Kontrolle(E)<br />
 +
-&gt;RV308
 +
Insert: 6&micro;l <br />
 +
vector: 3&micro;l <br />
 +
Ouick ligase buffer: 10&micro;l <br />
 +
Quick ligase: 1&micro;l <br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <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>
 +
<div style="margin-left: 40px;">
 +
<table style="text-align: left; width: 389px; height: 342px;"
 +
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>gele of the AGO pETb+ ssDNA cleavage assay. Lanes:
 +
Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<br />
 +
<br />
 +
</div>
 +
<ul>
 +
  <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> 11.10.09, Timo, Hannes, Max, Anika</h3>
<h3> 11.10.09, Timo, Hannes, Max, Anika</h3>
-
 
+
<br />
-
<br>* Digestion of
+
<ul>
-
1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI<br>
+
  <li>&nbsp;Digestion of&nbsp;</li>
-
2.pMAFos_HlsbZip, XbaI - AgI<br>
+
</ul>
-
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br>
+
<div style="margin-left: 40px;">1.pEXHisFluA (27.09.09,
-
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br>
+
Klon1), XbaI - PstI<br />
-
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br>
+
2.pMAFos_HlsbZip, XbaI - AgI<br />
-
6.pMA_BB057, XbaI - PstI (01.10.09)<br>
+
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br />
-
<br>*gel extraction of
+
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br />
-
- pMA<br>
+
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br />
-
- pEx<br>
+
6.pMA_BB057, XbaI - PstI (01.10.09)<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>gel extraction of
 +
- pMA</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- pEx<br />
- ...
- ...
-
 
+
<br />
-
<br>* Testdigestion of
+
</div>
-
7.pMA_YFP 2 (10.10.09, Caro)<br>
+
<ul>
-
8.pMA_CFP (10.10.09, Caro)<br>
+
  <li>&nbsp;Testdigestion of&nbsp;</li>
-
9.pMASplitLi1 (10.10.09, Caro)<br>
+
</ul>
-
10.pMASplitLi2 (10.10.09, Caro)<br>
+
<div style="margin-left: 40px;">7.pMA_YFP 2 (10.10.09,
-
...image..  
+
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.. <br />
-
<br>* plasmid prep of pEx_DsbA_StrepDigSplitFoka
+
</div>
-
<br>* plasmid prep of pEx_HisDigSplitFoka
+
<ul>
-
 
+
  <li>&nbsp;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>&nbsp;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>&nbsp;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>&nbsp;plasmid prep of pEx_DsbA_StrepDigSplitFoka
-
Enzymes: 1µl of NgoMIVI and 1.5µl PstI<br>
+
  </li>
-
Buffer: 3µl buffer 1<br>
+
  <li>&nbsp;plasmid prep of pEx_HisDigSplitFoka
-
BSA (100fold): 0.5µl<br>
+
  </li>
-
Water: 14.5µl<br>
+
  <li>&nbsp;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> 12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia,
-
7.pMA-CAT Ndelta4 (MQII)6<br>
+
Timo, Gerrit</h3>
-
8.pMAFoka clone 1 from 04.10.<br>
+
<br />
-
<br>*Made new BB-AGO PCR using digested AGO Gene without the vector-> still no expected bands to be seen...
+
<ul>
-
<br>*Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay  
+
  <li>&nbsp;protein expression of pEx_DsbA_StrepDigSplitFoka
-
<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
+
(periplasm) in
-
 
+
BL21de3 at 22&deg;C.
-
<br>*Send to sequencing:[[Protocols#DNA_Sequencing]]<br>
+
  </li>
-
Julia 1-6:<br>
+
  <li>&nbsp;made 5 litres of DYT
-
1)pBADQuick clone 3<br>
+
  </li>
-
2)pBAD T4 clone 2<br>
+
  <li>digest of pMASplitFoka clone 1 and 2 from 04.10.09
-
3)pBAD T4 clone 1<br>
+
DNA:10&micro;l
-
4)pMA YFP clone 1<br>
+
Enzymes: 1&micro;l of NgoMIVI and 1.5&micro;l PstI</li>
-
5)pMA CFP clone 1<br>
+
</ul>
-
6)pMA Split Linker clone 2<br><br>
+
<div style="margin-left: 40px;">Buffer: 3&micro;l
-
 
+
buffer 1<br />
 +
BSA (100fold): 0.5&micro;l<br />
 +
Water: 14.5&micro;l<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>&nbsp;Plasmidprep. of: </li>
 +
</ul>
 +
<div style="margin-left: 40px;">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.<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>Made new BB-AGO PCR using digested AGO Gene without the
 +
vector-&gt; 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;">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
-
 
+
<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>
-
->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
-
 
+
-&gt;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>
+
-&gt;analytical gel (see picture)</li>
-
into BL21 de3 gold<br>
+
</ul>
-
Plates: LB+ AMP +1%Glucose<br>
+
<div style="margin-left: 40px;">
-
<br>*cultivation of
+
-&gt;didn't work again<br />
-
pma strep dig split foka<br>
+
</div>
-
pma long linker<br>
+
<br />
-
pma his flua split foki<br>
+
<ul>
-
 
+
  <li>poured IPTG/XGal plates for in vivo plaque assay test run
-
 
+
  </li>
 +
  <li>transformation of:</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- pBAD_FokA (Quick)<br />
 +
- pBAD_CAT(Quick)<br />
 +
- pBAD_FokA (T4)<br />
 +
- pBAD_CAT (T4)<br />
 +
- pEX_FokA+YFP<br />
 +
- pEX_DsbA+FokA+YFP<br />
 +
into BL21 de3 gold<br />
 +
Plates: LB+ AMP +1%Glucose<br />
 +
</div>
 +
<br />
 +
<ul>
 +
  <li>cultivation of
 +
pma strep dig split foka</li>
 +
</ul>
 +
<div style="margin-left: 40px;">pma long linker<br />
 +
pma his flua split foki<br />
 +
</div>
<h3> 13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia</h3>
<h3> 13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia</h3>
-
 
+
<br />
-
<br>*Miniprep
+
<ul>
-
 
+
  <li>Miniprep
-
-pMA-fos 1<br>
+
-pMA-fos 1</li>
-
-pMA-fos 2<br>
+
</ul>
-
-pMA his flua split fokI<br>
+
<div style="margin-left: 40px;">-pMA-fos 2<br />
-
- pMA strep dig split foka <br>
+
-pMA his flua split fokI<br />
-
-pMA long linker<br>
+
- pMA strep dig split foka <br />
-
 
+
-pMA long linker<br />
-
<br>*testdigest of Plasmidprep from today
+
</div>
-
 
+
<br />
-
15 µl for 6µl loading dye
+
<ul>
-
-buffer 2 1µl<br>
+
  <li>testdigest of Plasmidprep from today
-
-xbaI 0.5µl<br>
+
15 &micro;l for 6&micro;l loading dye
-
-pstI 0.75µl<br>
+
-buffer 2 1&micro;l</li>
-
-DNA 2 µl<br>
+
</ul>
-
-water 9.75µl<br>
+
<div style="margin-left: 40px;">-xbaI 0.5&micro;l<br />
-
-BSA 1µl<br>
+
-pstI 0.75&micro;l<br />
-
 
+
-DNA 2 &micro;l<br />
-
--> Gelbild
+
-water 9.75&micro;l<br />
-
 
+
-BSA 1&micro;l<br />
 +
--&gt; 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)
+
<br />
-
->stored in -80°C
+
-
<br>*culture of ER2738 in LB Tet for cotransformation in afternoon
+
-
<br>*test in vivo assay
+
-
- electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV<br>
+
-
- 1.5h on 37°C shaker at 750rpm<br>
+
-
- precipitation of phages<br>
+
-
- infection of ER2738 with different dilutions of phages<br>
+
-
- mixed cells with Top agar and plated them on IPTG/XGAL plates -> 37° shaker<br>
+
-
<br>*inoculation of
+
-
-pMASplitFoka clone 1 from prep 04.10.09<br>
+
-
-pMAStrepDig clone 2 from prep 25.09.09<br>
+
-
-pMAMiddleFoka clone 2 from prep 04.10.09<br>
+
-
-pMAShortFoka clone 2 from prep 04.10.09<br>
+
-
-pMAFoka clone 1 from prep 04.10.09<br>
+
-
-pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09<br>
+
-
-pMALongFoka clone1 prep from 10.09.09<br>
+
-
-pMAFos 13.10.09<br>
+
-
-pBAD_CAT 13.10.09<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<br>
+
-
<br>*protein purification of DsbA_StrepDigSplitFoka with Strep column
+
-
 
+
-
<br>*digestion, 1% agarose gel and gel extraction of
+
-
- pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI<br>
+
-
- 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
+
-
- pMA (01.10., c=500ng/µl) with XbaI + AgeI
+
-
- pMA (01.10., c=500ng/µl) with EcoRI + SpeI
+
-
 
+
-
 
+
-
<br>*ligations:
+
-
Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI<br>
+
-
Insert: 6 µl LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br>
+
-
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><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>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br>
+
-
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<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 + PstI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
+
-
Insert: 6 µl Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
+
-
Insert: 6 µl Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><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 XbaI + AgeI<br>
+
-
Buffer: 10 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
+
-
Insert: 2 µl ShortLinker, hybridised, with XbaI + AgeI<br>
+
-
Buffer: 14 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
+
-
Insert: 2 µl MiddleLinker, hybridised, with XbaI + AgeI<br>
+
-
Buffer: 14 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
+
-
Insert: 2 µl LongLinker, hybridised, with XbaI + AgeI<br>
+
-
Buffer: 14 µl, QuickLigase Buffer NEB<br>
+
-
QuickLigase: 1 µl<br><br>
+
-
 
+
-
<br>*did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc
+
-
 
+
-
 
+
-
<br>*ELISA
+
-
-Blocking<br>
+
-
-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>
+
-
 
+
-
<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...
+
-
<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
+
-
<br>* Made ELISA with strep-biotinylated target oligo coated surface-> no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS
+
-
<br>* Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I
+
-
<br>* Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence
+
-
 
+
-
 
+
-
<br>
+
</div>
</div>
-
<div style="text-align: left;"><br />
+
<ul>
 +
  <li>made electrocompetent XL1 Blue cotransformed cells
 +
(pExHisFluASplitFoki and pJS419HisDigSplitFoka)
 +
-&gt;stored in -80&deg;C
 +
  </li>
 +
  <li>culture of ER2738 in LB Tet for cotransformation in
 +
afternoon
 +
  </li>
 +
  <li>test in vivo assay - electroporation with new
 +
electrocompetent cells
 +
and M13 ssDNA at 1.7kV</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- 1.5h on 37&deg;C
 +
shaker at 750rpm<br />
 +
- precipitation of phages<br />
 +
- infection of ER2738 with different dilutions of phages<br />
 +
- mixed cells with Top agar and plated them on IPTG/XGAL plates
 +
-&gt; 37&deg; shaker<br />
</div>
</div>
-
<div style="text-align: center;"><br />
 
<br />
<br />
 +
<ul>
 +
  <li>inoculation of
 +
-pMASplitFoka clone 1 from prep 04.10.09</li>
 +
</ul>
 +
<div style="margin-left: 40px;">-pMAStrepDig clone 2 from
 +
prep 25.09.09<br />
 +
-pMAMiddleFoka clone 2 from prep 04.10.09<br />
 +
-pMAShortFoka clone 2 from prep 04.10.09<br />
 +
-pMAFoka clone 1 from prep 04.10.09<br />
 +
-pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09<br />
 +
-pMALongFoka clone1 prep from 10.09.09<br />
 +
-pMAFos 13.10.09<br />
 +
-pBAD_CAT 13.10.09<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<br />
</div>
</div>
 +
<br />
 +
<ul>
 +
  <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;">- pMA-His-FluA-Split-FokI
 +
(13.10.) with SpeI + PstI<br />
 +
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI =&gt;
 +
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
 +
- pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI
 +
- pMA (01.10., c=500ng/&micro;l) with EcoRI + SpeI
 +
<br />
</div>
</div>
 +
<ul>
 +
  <li>ligations:
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with EcoRI +
 +
SpeI</li>
 +
</ul>
 +
<div style="margin-left: 40px;">Insert: 6 &micro;l
 +
LongLinker-FokA (10.09., clone 1) with EcoRI +
 +
SpeI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br />
 +
Insert: 6 &micro;l ShortLinker-FokA (04.10., clone 2) with NgoMIV +
 +
PstI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br />
 +
Insert: 6 &micro;l MiddleLinker-FokA (04.10., clone 1) with NgoMIV
 +
+ PstI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br />
 +
Insert: 6 &micro;l ShortLinker-FokA (04.10., clone 2) with NgoMIV +
 +
PstI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br />
 +
Insert: 6 &micro;l MiddleLinker-FokA (04.10., clone 1) with NgoMIV
 +
+ PstI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI +
 +
AgeI<br />
 +
Insert: 6 &micro;l Strep-FluA-MiddleLinker-FokI (08.10., clone 1)
 +
with XbaI + AgeI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI +
 +
AgeI<br />
 +
Insert: 6 &micro;l Strep-FluA-MiddleLinker-FokA (08.10., clone 1)
 +
with XbaI + AgeI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI +
 +
AgeI<br />
 +
Insert: 6 &micro;l Strep-FluA-LongLinker-FokI (08.10., Klon 1) with
 +
XbaI + AgeI<br />
 +
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI +
 +
AgeI<br />
 +
Insert: 2 &micro;l ShortLinker, hybridised, with XbaI + AgeI<br />
 +
Buffer: 14 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI +
 +
AgeI<br />
 +
Insert: 2 &micro;l MiddleLinker, hybridised, with XbaI + AgeI<br />
 +
Buffer: 14 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
<br />
 +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI +
 +
AgeI<br />
 +
Insert: 2 &micro;l LongLinker, hybridised, with XbaI + AgeI<br />
 +
Buffer: 14 &micro;l, QuickLigase Buffer NEB<br />
 +
QuickLigase: 1 &micro;l<br />
 +
</div>
 +
<br />
 +
<br />
 +
<ul>
 +
  <li>did transformations of the ligations in RV308, plated on
 +
LB-Amp + 1%
 +
Gluc
 +
  </li>
 +
  <li>ELISA -Blocking</li>
 +
</ul>
 +
<div style="margin-left: 40px;">-Washing with TBST-EDTA 5x<br />
 +
-Loaded immunotubes (1.5ml) and wells 100&micro;l each with 0.5mM
 +
biotin-target DNA<br />
 +
-Incubation at 20&deg;C in shaking<br />
 +
-Incubatet Phages with Oligo for 30min at 55&deg;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&deg;C<br />
 +
-Loaded immunotubes (each 1.5 ml) with 50&micro;l P1,
 +
25&micro;l 449Phage+ Oligo in TBST-EDTA<br />
 +
-Incubation in shaker at 20&deg;C<br />
 +
-Washing of wells and immunotubes with TBST-EDTA 5x
 +
-Loaded 100&micro;l/well anti-M13 ab(1:1000), incubated for 1 hour
 +
at 20&deg;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 />
 +
</div>
 +
<br />
 +
<ul>
 +
  <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-&gt; 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-&gt; 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-&gt; 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>
 +
</ul>
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<br />
<br />
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+
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 +
<div class="art-Footer">
 +
<div class="art-Footer-inner">
 +
<div class="art-Footer-text">
 +
<p>contact:&nbsp; <a
 +
href="mailto:freigem09@googlemail.com">freigem09@googlemail.com</a><br />
 +
</p>
</div>
</div>
</div>
</div>
 +
<div class="art-Footer-background"></div>
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Revision as of 19:19, 20 October 2009

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> FREiGEM

  October

01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel


  •  Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
  •  Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
  •  plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
  •  plasmid preparation of pEX-Strep-Dig-Split-Fok(active) Klon 1
  •  glycerin stock of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 (RV308), stored at -80°C

  • protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column
  • 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]]
  • pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4)
  • phage display: - desalt ligation products (for electroporation)
pool samples (~80µl 445+87/89 and ~52µl 445+87/88)
add 1 volume of isopropanol, mix
-80°C for 10 minutes
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant
add 1 volume of 75% ethanol (without mixing)
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant
let dry on heat block (50°C), lid open
add 25µl water
thermo shaker for 1h, 45°C, 1000rpm - PCR (4 samples each template):
buffer (with MgCl2): 5µl
primer #7: 1,5µl
primer #1: 1,17µl
dNTPs: 2µl
Taq: 1µl
MnCl2: 0,5µl
MgCl2: 5µl
water: 33µl
DNA (425 or 428): 5µl
- PCR:
DNA templates: pJs#448 (0,3µl), pJs#449 (0,3µl), pJs#375 (1µl), pJs#413 (0,3µl), pJs#445 (0,3µl)
primers (#95, #7): 1,5µl
high fidelity buffer: 5µl
dNTPs: 1µl
TMenzyme: 0,3µl
water: 39,7µl

  •  Overnight Culture RV308 for Competent Cells, on shaker in 37 °C room
  • analysis of sequences from 28.09.09
  • digest of pExStrepDigSplitFoka prep from 30.09.09 Plasmid: 15 µl
water: 9 µl
BSA: 0.5 µl NEB iGEM stock
buffer: 3 µl buffer 3 NEB iGEM stock
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock

  • digest of PCR product RBSStrepDigSplitFoka from 30.09.09 Plasmid: 10 µl
water: 14 µl
BSA: 0.5 µl NEB iGEM stock
buffer: 3 µl buffer 3 NEB iGEM stock
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock

*digest of pExHisFluaSplitFoki prep from 24.08.09 Plasmid: 15 µl
water: 9 µl
BSA: 0.5 µl NEB iGEM stock
buffer: 3 µl buffer 2 NEB iGEM stock
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock
-> put in 37°C room for 2h ->preparative gel

Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka


 interpretation: bands had right size but pExStrepDigSplitFoka showed unexpectedly low concentration
->gelextraction
ligation of
- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)

  • Inoculation of - pJS419_StrepDigSplitFoka
- pJS419_HisDigSplitFoka
- pEx_HisFluA
- pEx_HisDigMiddleLiFoka
  • digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
  • Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09 --> make glycerol stocks tomorrow
  • agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
  • cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more)
  • gel slices stored at -4°C over night

02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max


  • phage display - OD600 of the pre-culture should be 0,2
- 37°C shaker
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)
- Apportion culture into 10x50 ml Falkon tubes
- 15 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)
- resuspend pellets in 25 ml H2O (10-> 8 tubes)
- 15 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4 tubes)
- 15 minutes on ice
- Centrifuge 10 min at 4°C / 2500
- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-> 2 tubes)
- 5 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)
- 5 minutes on ice
- Centrifuge 10 min at 4°C / 2500 g
- Discard supernatant, pool pellets in 1 ml 10% DMSO
- Measure OD600 (1:100 dilution), OD should be 0,4
- Make aliquots à 80 µl, souse with N2
- Store at -80°C

  • glycerol stock of pEx_HisFluASplitFoki in BL21de3
  • SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09 --> do a Western Blot
  • new ampicilin 100 aliquots
  • preparation of M13dsDNA
  • chem. competent cells aliqots
  • Periplasma Project Digestion:
- digested with xba and spe --> GelBilder

  •  Gelextraction Min Elute Gel Extraction Kit
- mesure mass: StrepFokA = 50 mg - JS418= 90mg
-JS419=80mg
- HisFokA = 250mg
Nandropdata
  • Ligation - with Quickligase JS119-StrepFokA
JS118-Strep
JS118-HisFokA
JS19-HisFokA
- 15 min at 25°C
  • Transformation - 2YT-Medium 950 µL to 50µl cells and DNA
BL21-JS190
BL21-JS119StrepFokA
BL21-JS118Strep
BL21-JS118HisFokA
BL21-JS19HisFokA - Plated on KM plats
  •  Preparation of overnight cultures 5 ml LB medium each - pBad+Kan
-pET39B(+)
  • Amp
--> Stored in 37°C room
  • Digest: - pMASplitFoka
- pMAFoka
- pMAFoki
- pMASplitFoki
- pMALongLiFoka
- pMALongliFoki
- pMAShortli
- pMAMiddleli
- pMALongli
- pExStrepFluA
  • preparative gel [[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]] ->interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly
  • gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09
  • digest and ligation into new pMA:
-pMASplitFokA clone1 19.08.09
-pMAFokA clone2 05.08.09
-pMAFoki clone2 05.08.09
-pMASplitFoki clone2 06.08.09
-pMAshortLi clone2 10.09.09
-pMAmiddelLi clone2 10.09.09
-pMAlongLi clone1 10.09.09
-pMAlongLiFokA clone1 10.09.09
-pMAlongLiFoki clone1 10.09.09
digestion with EcoRI and SpeI (vector and insert)

  •  Transformation of:
-pMAFokA clone2 05.08.09
-pMAFoki clone2 05.08.09
-pMASplitFoki clone2 06.08.09
  • plasmidprep of:
1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick
2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick
3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick

4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick
5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick
6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick

7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit1
8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit2
9. pEX_DsbA+Strep+Dig+Split+FokA Clone3

10. pEX_DsbA+His+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit3
11. pEX_DsbA+His+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit4
12. pEX_DsbA+His+Dig+Split+FokA Clone3
  • testdigest of: V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick
V2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick
V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick

V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick
V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick
V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick

V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1
V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2
V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3

V10. pEX_DsbA+His+Dig+Split+FokA Clone1
V11. pEX_DsbA+His+Dig+Split+FokA Clone2
V12. pEX_DsbA+His+Dig+Split+FokA Clone3

V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)
V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)
with NcoI-HF and XbaI 1h at 37°C
--> geldbild

03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph


  • plasmid prep of pET and pBad and Glytsocks(Xbl)
  • new aliquots Ampicilin (70%EtOH)
  • 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]]
  • digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)
- pEx-Strep-Dig-MiddleLinker-Fok(inactive)
- pEx-Strep-Dig-ShortLinker-Fok(inactive)
- pEx-Strep-Dig-SplitLinker-Fok(inactive)
- pEx-His-Dig-SplitLinker_Fok(inactive)
- pEx-His-Dig-SplitLinker_Fok(active)
- pEx-Strep-Dig-SplitLinker-Fok(active)
- pEx-His-Dig-LongLinker-Fok(inactive)
- pEx-His-FluA-LongLinker-Fok(inactive)
- pEx-His-FluA-MiddleLinker-Fok(inactive)
- pEx-His-FluA-ShortLinker-Fok(inactive)
- pEx-His-FluA-SplitLinker-Fok(inactive)
- pEx-His-Dig
- pMA
with NgoMIV and SpeI
  • digest of - pMA with XbaI and AgeI
  • Ligation of: - Strep-Dig-LongLinker-Fok(inactive)
- Strep-Dig-MiddleLinker-Fok(inactive)
- trep-Dig-ShortLinker-Fok(inactive)
- Strep-Dig-SplitLinker-Fok(inactive)
- His-Dig-SplitLinker_Fok(inactive)
- His-Dig-SplitLinker_Fok(active)
- Strep-Dig-SplitLinker-Fok(active)
- His-Dig-LongLinker-Fok(inactive)
- His-FluA-LongLinker-Fok(inactive)
- His-FluA-MiddleLinker-Fok(inactive)
- His-FluA-ShortLinker-Fok(inactive)
- His-FluA-SplitLinker-Fok(inactive)
into pMA
and
- Strep-Dig-LongLinker-Fok(inactive)
- Strep-Dig-MiddleLinker-Fok(inactive)
- trep-Dig-ShortLinker-Fok(inactive)
into new pEX
  • inoculation of: -pMASplitFokA clone1 19.08.09
-pMAFokA clone2 05.08.09 (new)
-pMAFoki clone2 05.08.09 (new)
-pMASplitFoki clone2 06.08.09 (new)
-pMAshortLi clone2 10.09.09
-pMAmiddelLi clone2 10.09.09
-pMAlongLi clone1 10.09.09
-pMAlongLiFokA clone1 10.09.09
-pMAlongLiFoki clone1 10.09.09

-pMAFokA clone2 05.08.09 (old)
-pMAFoki clone2 05.08.09 (old)
-pMASplitFoki clone2 06.08.09 (old)

  • inoculation and plasmid preperation: -pEX_strepFluA
  • Quenching test with HisFluASplitFoki and fluorescin-tagged oligos
  • BB Ago Pcr via Taq
  • Started makin new VCS M13 Phages see Protocol day 1

Plan for 04.10.09


  • inoculation of - pMA-Strep-Dig-LongLinker-Fok(inactive)
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)
- pMA-Strep-Dig-ShortLinker-Fok(inactive)
- pMA-Strep-Dig-SplitLinker-Fok(inactive)
- pMA-His-Dig-SplitLinker_Fok(inactive)
- pMA-His-Dig-SplitLinker_Fok(inactive)
- pMA-Strep-Dig-SplitLinker-Fok(active)
- pMA-His-Dig-LongLinker-Fok(inactive)
- pMA-His-FluA-LongLinker-Fok(inactive)
- pMA-His-FluA-MiddleLinker-Fok(inactive)
- pMA-His-FluA-ShortLinker-Fok(inactive)
- pMA-His-FluA-SplitLinker-Fok(inactive)
into LB-Amp
  • digestion of pEx-Strep-Flua with AgeI and PstI
  • digestion of pMA-Short/Middle/Long/Split-Fok(active and inactive)
  • ligation of the above parts and transformation into RV308
  • plasmid preparation of... -pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLi
-pMAmiddelLi
-pMAlongLi
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)

04.10.09, Laura, Anika, Max


  •  Plasmid preparation of -pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 1 and 2,respectively - results see notebook
  •  Glycerolstock of
-pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 3
  •  Pellets of
-pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 1 and 2,respectively - stored in Pelletbox, -20°C
  • Digest of 1. pEXHisDigMiddleLFoki 2. pMaScFaantiNIP 3. prep of Ligation pEX+CAT of 09.07.09 old 4. pMAShortLFoka
5. pMAShortLFoki
6. pMAMiddleLFoka
7. pMAMiddleLFoki
8. pMALongLFoka
9. pMALongLFoki
10.pEXStrepFlua

  • Preparative Gel of Digest
Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA


  • Gelextraction
  • Ligation of
1.pMAHisDigMiddleFoki
2.pMACAT
3.pEXStrepFluAShortLFokA
4.pEXStrepFluAShortLFoki
5.pEXStrepFluAmiddleLFokA
6.pEXStrepFluAMiddleLFoki
7.pEXStrepFluALongLFokA
8.pEXStrepFluALongLFoki
9.JS 419StrepDigSplitFokA
10.JS 419HisDigSplitFokA
11.JS 418HisDigSplitFokA
Approach: 8µl H2O, 3 µl Vector, 6µl Insert, 2µl Quick Ligase Buffer, 1 µl Quick Ligase, 15 min, Room Temp.

  • Dephosphorylation of pBAD Vector:
Approach: 2µl Eluat, 3,1 µl Fast Ap Buffer 10x, 1µl Fast Ap, 10 min. 37°C, 5 min. 75°C

  • Transformation of
1.pMAHisDigMiddleFoki (RV)
2.pMACAT(RV)
3.pEXStrepFluAShortLFokA(RV)
4.pEXStrepFluAShortLFoki(RV)
5.pEXStrepFluAmiddleLFokA(RV)
6.pEXStrepFluAMiddleLFoki(RV)
7.pEXStrepFluALongLFokA(RV)
8.pEXStrepFluALongLFoki(RV)
9.pBAD (RV)
10.pJS 419StrepDigSplitFokA(XBL)
11.pJS 419HisDigSplitFokA(XBL)
12.pJS 418HisDigSplitFokA(XBL)

  • test digest of plasmid preps from today -> run on a gel with digest pET39, xbaI, ecoRI from Tobi
  • starter culture of pEx_DsbA_HisDigSplitFoka -> modified expression has to be done tomorrow
  • inoculation of pJS418 and pJS419 for glycerolstocks tomorrow
  • Taq AGO BB PCR did not work
  • Phageproduction day 2

05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika


  •  3l LB and 2L LB Agar
  • Periplasma Project
  • digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09)
  • test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka
  • 1) Gelextraction
--> Gelbild
1) Vector: pEXHisFluSplitFoki
2)Insert: RBSStrepDigSplitFokA
3)PET39b+

  • 2)Ligation
-10ul 2 fold buffer
- 6 µl Insert
-3µl Vector
-1µl Quick Ligase
--> 15 min at RT
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA

  • 3)Transformation
-5 µl DNA of Ligation + BL21d3 and XBL
  • '''Mini prep with with Qiagen Spin Miniprep Kit of''' - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3
- pMA-Dbsa 1,2,3
  • '''Made Glycerolstocks of'''
  • 700µl Cellsuspension+ 300µl Glycerol - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(active)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3
- pMA-Dbsa 1,2,3
  • '''Test Digestion of'''
  • put in each sample: 5µl DNA, 2,5µl H2O, 1µl Buffer 3, 1µl EcoRI, 1µl PstI, 1µl BSA - pMA-Strep-Dig-LongLinker-Fok(inactive)3
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1
- pMA-Strep-Dig-SplitLinker-Fok(inactive)3
- pMA-His-Dig-SplitLinker_Fok(inactive)1
- pMA-His-Dig-SplitLinker_Fok(active)2
- pMA-Strep-Dig-SplitLinker-Fok(active)1
- pMA-His-Dig-LongLinker-Fok(inactive)1
- pMA-His-FluA-LongLinker-Fok(inactive)3
- pMA-His-FluA-MiddleLinker-Fok(inactive)3
- pMA-His-FluA-ShortLinker-Fok(inactive)1
- pMA-His-FluA-SplitLinker-Fok(inactive)2
- pMA-Dbsa 2

  •  Transformation of
- XBL pEXHisFluASplitFokIRBSStrepDigSplitFokA
- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA

  • Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each - induced with 0,7mM IPTG and took samples T0-T5
- centrifuged in buckets 17', 4000rpm, 4°C
- eluted in 20 ml TES in each bucket
....
  • PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -> very few product, as well as non specific ones
  • PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature

06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika


  • digest - pBAD vector
DNA: 16.8µl
enzyme: 1µl AgeI
buffer: 2µl buffer 1
- pBAD insert
DNA: 16.8µl
enzyme: 1µl XmaI
buffer: 2µl buffer 4
BSA (10X): 2µl
- pJS 418/419
DNA: 10µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 3µl buffer 3
BSA (10X): 3µl
- pExStrepDigSplitFoka/pExHisDigSplitFoka
DNA: 16.8µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 3µl buffer 3
BSA (10X): 3µl

  • testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA: 5µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 1µl buffer 3
BSA (10X): 1µl


  • preparative gels of digests
preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418 preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka
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
  • testdigest of pMAdsba DNA: 5µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 1µl buffer 3
BSA (10X): 1µl

  • 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
  • Ligation - Dephphorylation of pBAD Gelex
- 1µl Fast AP
- 5.5µl fast AP buffer
-1.5µl water
--> Solution was given to eluat - for each ligation:
- 6µl Insert
-3µl Vetor
-1µl Quickligase
-10µl buffer
--> pBAD
  •  Insert (Dummy)
--> pJS419+HisDigSplitFoka -->pJ418+HisDigSplitFoka
  •  Transformation --> pBAD
  •  Insert (Dummy)
--> pJS419+HisDigSplitFoka -->pJ418+HisDigSplitFoka - in XLblue
-pBAD was plated on AMP plates
- Both pJS... were plated on CM plates

  • Ligation and Transformation of
 Ligation:per sample 8µl H2O; 3µl vector; 6µl insert; 2µl Quick Ligase buffer; 1µl Quick Ligase Transformation:Defrost competent cells on ice:(100 µl); add of the ligation: 5 µl; DNA and cells: mix softly by knocking; Incubation on ice for: 20-30 min; Heat shock at: 42°C for 40 sec; Cool off on ice for: 5 min; add sterile LB(or dyt)medium: 900 µl; Incubation in(shaker)at: 37°C for 60-70 min; Plate cells on LB+antibiotic plates: ampicillin; ligation: 2 plates: 1. 50µl cells 2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the restcells and plate out: -pMA HisDig Middle Linker Foki

-pMA CAT
-pEX Strep FluA SL FokA
-pEX Strep FluA LL Foki
-pEX Strep FluA ML Foki
-pEX Strep FluA ML Foka
-pEX Strep FluA LL Foka

  • Sequencing: 27µl H2O; 3µl DNA -pMA Dbsa clone 2
-pMA HisDigSplitFokA clone 2
-pMA HisFluASL Foki clone 1
-pMA HisFluASplitFoki clone 2
-pMA StrepDigLLFoki clone 3
-pMA HisFluaMLFoki clone 3
-pMA StrepDigSplitFokA clone 1
-pMA StrepDigMLFoki clone 1
-pMA StrepDigSLFoki clone 2
-pMA HisDigLLFoki clone 1
-pMA StrepDigSplitFoki clone 3
-pMA HisDigSplitFoki clone 1
-pMA HisFluaLLFoki clone 3

  • Inoculation of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room overnight
  • 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)
  • 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
  • Started dialysis to transfer the leftover AGO-proteins into the assay buffer

07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika


  • 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]]
  •  Inoculation 4 Clones respectively:
- pMA-His-Dig-MiddleLinker-Foki in XLBlue
- pJS419-HIs-Dig-Split-Foka- in XLBlue
- pJS418-HIs-DIg-Split-Foka- in XLBlue
- pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest
- pEX-Strep-FluA-LongLinker-Foki in XL1blue rest
- pMA-CAT in XLblue 10µl
-pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest
2 Clones respectively:
-pEX-His-FluA-Split-Foki + Glycerolstock vom 26.08.09 - pEX-Strep-Dig-Split-Foka + Glycerolstock vom 29.08.09
  •  Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3
  • two step PCR assembly of DsbA, His_Fos, and SplitFoka - program name: Assembly
- three different samples: 1. without DMSO, 2. with DMSO, 3. without DMSO and with last primers just added after first step
  • preparative gel of the PCR samples -> primer haven't been diluted an probably made all secondary structures, has to be repeated
  • digest of pBAD with AgeI and new PCR with insert digested with XmaI
  • preparative gel with digest and
  • test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka DNA: 5µl
Enzymes: 0.5µl of BamHI and MfeI each
Buffer: 1µl buffer 4
BSA (10fold): 1µl
Water: 2µl
-> pJS419_HisDigSplitFoka showed bands of the right size and was sequenced with primer sf_lac P1

  • plasmidpreparation of - pEXHisFluASplitFoki
- pExStrepDigSplitFoka
-> low concentrations

  • digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl
Enzymes: 1µl of PstI and 1.5µl SpeI
Buffer: 3µl buffer 2
BSA (100fold): 0.5µl
Water: 9µl
pExStrepDigSplitFoka: 15µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 9µl
pExStrepDigSplitFoka: 10µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 14µl

  •  Gelextraction of digest 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst)
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)

  • preparative gels of digests
[[Image:|none|thumb|preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product]] [[Image: |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
RBS PCR Product: m= 90 mg
vector: 0 0 130 mg

  •  Ligation - 6µl Insert
-3µl Vetor
-1µl Quickligase
-10µl buffer
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka

  • Transformation - All in XL1blue
- Vector: pEX-strep-Duig-Split-Foka
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka

  • 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
  • prepaired ELISA with anti-flag antibodies
  • made electro competent cells for transformation with the ago phagmidbibliothek

08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes


  • Poured SDS-Gels
  • Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet
-Transfomation with 3µl M13 Phage stock (-80°) at 10:30Uhr , incubated for 4-5 hours at 37°C in shaker
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml precipitation over night on ice in -4°C room

  • Miniprep - 2 clones respectively:
-A = pEX strep dig split foka
  • Finished Phage production(see protocol day 2): We obtained approximately 3.8
  • 10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1
  • 10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of them to the anti-Flag ELISA

  • Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):
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.

  1 2 3 4 5 6 7 8 9 10 11 12
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
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
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
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
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
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
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
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
     

  •  Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction
  • digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl
Enzymes: 1µl of PstI and 1.5µl SpeI
Buffer: 3µl buffer 2
BSA (100fold): 0.5µl
Water: 9µl
pExHisDigSplitFoka: 15µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 14µl
RBSStrepDigSplitFoka: 5µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 19µl
->made two digest of PCR construct

  •  Gelextraction of digest 1) Insert: His-Dig-Split-Foka( digested with xba and pst)
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)
and also PCR purification of RBS-Strep-Dig-Split-FokA
  • preparative gels of digests ->see picture

  • ligation of -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from gelex)
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR purification)

  • transformation of ligations
  • inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol
  • new two step PCR with all three plasmids for Fos construct (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution and preparative agarose gel ->interpretation: no band of 921bp was visible

  • one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel ->see picture
->interpretation: pMAFos and pMASplitFoka showed bands of the right size (230bp and 657bp respectively), of pMADsbA no product was visible

  • new one step PCR of pMADsba with newly prepared 1:1000 dilution
  • test digest of plasmidpreparations from today ->see picture

09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika


  • Phage ss DNA
-Over night in 4°C room precipitated phages: centrifuged for 20 min at 5000rpm and 4°C
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet recognized)
-Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at 13000rpm
-Decant the supernatant into new eppis, precipitate with 170µl PEG/NaCl and leave for 1 hour on ice

  • Miniprep - pJS 419-his-dig-split-foka (clon 1+2)
- Glycerolstock of clon1 and 2
- 300µl Glycerol
- 700 µl culture
- Pellets from clones 3,5,6
Nanodrop data:
pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl
pJS 419-his-dig-split-foka-clon2 =468 ng/µl

  • Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room
  • send to sequencing:
pMA Dig Plasmidprep 22.07.09 Timo1
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3
pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4
pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5
pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6
pMA Cat plasmidprep clone1 08.10.09 Timo7
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12

  •  concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation
  •  electrical trafo of the 449 ago phagmid bibliothek into XL1
  •  prepaired immutubes with streptavidin for ago phages test panning tomorrow

10.10.09 Julia, Caro, Laura, Christoph


  • 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
  • Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM
  • 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
  • Decanted to 16x50ml falcon tubes, centrifuged for 20min at 5000rpm and 4°C, decanted supernatant into new 16x50ml falcons with each 7ml PEG/NaCl
  • Precipitaion over night in 4°C room
  • plasmidpreparation of - pBADQuick clones 1-6
- pBADT4 clones 1-6
- pMAYFP clone 1-2
- pMACFP clone 1
- pMASplitlinker clones 1-2
- HisTag clones 1-2 (but test digest was negative -> thrown away)
<
  • 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
Enzymes: 1µl of NgoMIVI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (10fold): 3µl
Water: 10,5µl
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
Enzymes: 1µl of AgeI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (10fold): 3µl
Water: 10,5µl

  • PCR assembly with pMADsbA, different approaches (Taq Polymerase or Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and pMAFos together
  • analytical gel of PCRs -> didn't work
  • preparative gels of digests -> see picture -> no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more
  • ligation of -pMAStrepDigShortLiFoka
-pMAStrepDigMiddleLiFoka
-pMAStrepDigLongLiFoka
-pExStrepDigShortLiFoka
-pExStrepDigMiddleLiFoka
-pExStrepDigLongLiFoka
-pMAHisDigShortLiFoka
-pMAHisDigMiddleLiFoka
-pMAHisDigLongLiFoka
-pMACATNd4 (MQI)
-pMACATNd4 (MQII)
-pMA Kontrolle (M)
-> in XLBlue
- pEx_CATNd4 (EQI)
- pEx_CATNd4 (EQII)
- pEx_Kontrolle(E)
->RV308 Insert: 6µl
vector: 3µl
Ouick ligase buffer: 10µl
Quick ligase: 1µl

  • transformation of ligations in XBL on LB/Amp/1%glucose plates
  • cotransformation of
  • testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture
gele of the AGO pETb+ ssDNA cleavage assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA


  • cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates
  • inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09

11.10.09, Timo, Hannes, Max, Anika


  •  Digestion of 
1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI
2.pMAFos_HlsbZip, XbaI - AgI
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI
6.pMA_BB057, XbaI - PstI (01.10.09)

  • gel extraction of - pMA
- pEx
- ...
  •  Testdigestion of 
7.pMA_YFP 2 (10.10.09, Caro)
8.pMA_CFP (10.10.09, Caro)
9.pMASplitLi1 (10.10.09, Caro)
10.pMASplitLi2 (10.10.09, Caro)
...image..
  •  1% Agarosegel of constructs above
  •  Phage breeding day 2
  •  Starter culture of pEx_DsbA_StrepDigSplitFoka (Bl21de3)
  •  plasmid prep of pEx_DsbA_StrepDigSplitFoka
  •  plasmid prep of pEx_HisDigSplitFoka
  •  M13 ssDNA produced with bacterial of Julia and tried another variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected withM13phage particles and let it grow for 2h. After this followed the qiagen M13 protocol for M13Dna

12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia, Timo, Gerrit


  •  protein expression of pEx_DsbA_StrepDigSplitFoka (periplasm) in BL21de3 at 22°C.
  •  made 5 litres of DYT
  • digest of pMASplitFoka clone 1 and 2 from 04.10.09 DNA:10µl Enzymes: 1µl of NgoMIVI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (100fold): 0.5µl
Water: 14.5µl

  •  Plasmidprep. of:
1.pMAFokA (old)
2.pMA-CAT Ndelta4 (MQII)1
3.pMA-CAT Ndelta4 (MQII)2
4.pMA-CAT Ndelta4 (MQII)3
5.pMA-CAT Ndelta4 (MQII)4
6.pMA-CAT Ndelta4 (MQII)5
7.pMA-CAT Ndelta4 (MQII)6
8.pMAFoka clone 1 from 04.10.

  • Made new BB-AGO PCR using digested AGO Gene without the vector-> still no expected bands to be seen...
  • Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay
  • 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
  • Send to sequencing:[[Protocols#DNA_Sequencing]]
Julia 1-6:
1)pBADQuick clone 3
2)pBAD T4 clone 2
3)pBAD T4 clone 1
4)pMA YFP clone 1
5)pMA CFP clone 1
6)pMA Split Linker clone 2

Gerrit1: pMA_cat-Nd4
  • Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for Panning Simulation for tomorrow
  • made chemical competent cells of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki ->will prepare electrocompetent cells tomorrow
  • inoculation of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp
  • PCR of pMADsba repeated with different (higher) template dilutions ->analytical gel (see picture)
->didn't work again

  • poured IPTG/XGal plates for in vivo plaque assay test run
  • transformation of:
- pBAD_FokA (Quick)
- pBAD_CAT(Quick)
- pBAD_FokA (T4)
- pBAD_CAT (T4)
- pEX_FokA+YFP
- pEX_DsbA+FokA+YFP
into BL21 de3 gold
Plates: LB+ AMP +1%Glucose

  • cultivation of pma strep dig split foka
pma long linker
pma his flua split foki

13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia


  • Miniprep -pMA-fos 1
-pMA-fos 2
-pMA his flua split fokI
- pMA strep dig split foka
-pMA long linker

  • testdigest of Plasmidprep from today 15 µl for 6µl loading dye -buffer 2 1µl
-xbaI 0.5µl
-pstI 0.75µl
-DNA 2 µl
-water 9.75µl
-BSA 1µl
--> Gelbild - Glycerolstocks are in in box of 5/10/2009
  • made electrocompetent XL1 Blue cotransformed cells (pExHisFluASplitFoki and pJS419HisDigSplitFoka) ->stored in -80°C
  • culture of ER2738 in LB Tet for cotransformation in afternoon
  • test in vivo assay - electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV
- 1.5h on 37°C shaker at 750rpm
- precipitation of phages
- infection of ER2738 with different dilutions of phages
- mixed cells with Top agar and plated them on IPTG/XGAL plates -> 37° shaker

  • inoculation of -pMASplitFoka clone 1 from prep 04.10.09
-pMAStrepDig clone 2 from prep 25.09.09
-pMAMiddleFoka clone 2 from prep 04.10.09
-pMAShortFoka clone 2 from prep 04.10.09
-pMAFoka clone 1 from prep 04.10.09
-pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09
-pMALongFoka clone1 prep from 10.09.09
-pMAFos 13.10.09
-pBAD_CAT 13.10.09
-pBAD_Foka 13.10.09
-pMADsbAHisDigSplitFoka 13.10.09
-pMADsbAStrepDigSplitFoka 13.10.09
-pExFosSplitFoka 13.10.09
-pMAFosSplitFoka 13.10.09

  • protein purification of DsbA_StrepDigSplitFoka with Strep column
  • digestion, 1% agarose gel and gel extraction of - pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI
- pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI => wasn't ok on the gel, inoculated clone 1
- pMA-ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
- pMA-MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
- pMA-His-Dig (24.08., clone 2) with AgeI + PstI
- pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
- pEx-Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI
- pEx-Strep-FluA-MiddleLinker-FokA (08.10., clone 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 EcoRI + SpeI
  • ligations: Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI
Insert: 6 µl LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI
Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 6 µl Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 6 µl Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 6 µl Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 2 µl ShortLinker, hybridised, with XbaI + AgeI
Buffer: 14 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 2 µl MiddleLinker, hybridised, with XbaI + AgeI
Buffer: 14 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 2 µl LongLinker, hybridised, with XbaI + AgeI
Buffer: 14 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl


  • did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc
  • ELISA -Blocking
-Washing with TBST-EDTA 5x
-Loaded immunotubes (1.5ml) and wells 100µl each with 0.5mM biotin-target DNA
-Incubation at 20°C in shaking
-Incubatet Phages with Oligo for 30min at 55°c waterbath
-Washing of wells and immunotubes with TBST-EDTA 5x
-Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1 phage+Oligo and 1+control as a control
-Incubation in shaker at 20°C
-Loaded immunotubes (each 1.5 ml) with 50µl P1, 25µl 449Phage+ Oligo in TBST-EDTA
-Incubation in shaker at 20°C
-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
-Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS 3x
-2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS-T 3x
-3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis

  • 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...
  • AGO assay using different targets-> were not able to reproduce the presumed cutting event from Saturdays assay probably due to the lower DNA concentration
  • Made ELISA with strep-biotinylated target oligo coated surface-> no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS
  • Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I

contact:  freigem09@googlemail.com

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