Team:Freiburg bioware/Notebook/October

From 2009.igem.org

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October<span class="art-PostHeader"></span> </h2>
October<span class="art-PostHeader"></span> </h2>
</div>
</div>
-
<h5>
+
<h3 style="margin-left: 40px;">01.10.09, Hannes, Manu,
-
<h3>01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph,
+
Laura, Gerrit, Sarah, Christoph,
Caro, Julia, Anika, Isabel</h3>
Caro, Julia, Anika, Isabel</h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Tetracycline; 10 x 1ml, Concentration: 25 g/l,
   <li>&nbsp;Tetracycline; 10 x 1ml, Concentration: 25 g/l,
stored in -20&deg;C, box with antibiotics
stored in -20&deg;C, box with antibiotics
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   </li>
   </li>
</ul>
</ul>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>protein purification of HisFluASplitFoki (expressed in
   <li>protein purification of HisFluASplitFoki (expressed in
BL21de3) with NiNTA column
BL21de3) with NiNTA column
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- desalt ligation products (for electroporation)</li>
- desalt ligation products (for electroporation)</li>
</ul>
</ul>
-
pool samples
+
<div style="margin-left: 40px;"><small>pool samples
-
(~80&micro;l 445+87/89 and ~52&micro;l 445+87/88)<br />
+
(~80&micro;l 445+87/89 and ~52&micro;l 445+87/88)</small><br />
-
add 1 volume of isopropanol, mix<br />
+
<small>add 1 volume of isopropanol, mix</small><br />
-
-80&deg;C for 10 minutes<br />
+
<small>-80&deg;C for 10 minutes</small><br />
-
centrifuge for 10 minutes at max rpm, 4&deg;C; discard supernatant<br />
+
<small>centrifuge for 10 minutes at max rpm, 4&deg;C; discard
-
add 1 volume of 75% ethanol (without mixing)<br />
+
supernatant</small><br />
-
centrifuge for 10 minutes at max rpm, 4&deg;C; discard supernatant<br />
+
<small>add 1 volume of 75% ethanol (without mixing)</small><br />
-
let dry on heat block (50&deg;C), lid open<br />
+
<small>centrifuge for 10 minutes at max rpm, 4&deg;C; discard
-
add 25&micro;l water<br />
+
supernatant</small><br />
-
thermo shaker for 1h, 45&deg;C, 1000rpm
+
<small>let dry on heat block (50&deg;C), lid open<br />
-
- PCR (4 samples each template):<br />
+
<small>add 25&micro;l water</small><br />
-
buffer (with MgCl2): 5&micro;l<br />
+
<small>thermo shaker for 1h, 45&deg;C, 1000rpm
 +
- PCR (4 samples each template):</small><br />
 +
<small>buffer (with MgCl2): 5&micro;l</small><br />
primer #7: 1,5&micro;l<br />
primer #7: 1,5&micro;l<br />
primer #1: 1,17&micro;l<br />
primer #1: 1,17&micro;l<br />
Line 188: Line 192:
dNTPs: 1&micro;l<br />
dNTPs: 1&micro;l<br />
TMenzyme: 0,3&micro;l<br />
TMenzyme: 0,3&micro;l<br />
-
water: 39,7&micro;l<br />
+
water: 39,7&micro;l</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Overnight Culture RV308 for Competent Cells, on
   <li>&nbsp;Overnight Culture RV308 for Competent Cells, on
shaker in 37 &deg;C room
shaker in 37 &deg;C room
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Plasmid: 15 &micro;l</li>
Plasmid: 15 &micro;l</li>
</ul>
</ul>
-
water: 9 &micro;l<br />
+
<div style="margin-left: 40px;"><small>water: 9
 +
&micro;l<br />
BSA: 0.5 &micro;l NEB iGEM stock<br />
BSA: 0.5 &micro;l NEB iGEM stock<br />
buffer: 3 &micro;l buffer 3 NEB iGEM stock<br />
buffer: 3 &micro;l buffer 3 NEB iGEM stock<br />
Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock<br />
Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock<br />
Restriction_enzyme_2 : 1 &micro;l PstI from NEB KuKlabstock <br />
Restriction_enzyme_2 : 1 &micro;l PstI from NEB KuKlabstock <br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>digest of PCR product RBSStrepDigSplitFoka from 30.09.09
   <li>digest of PCR product RBSStrepDigSplitFoka from 30.09.09
Plasmid: 10 &micro;l </li>
Plasmid: 10 &micro;l </li>
</ul>
</ul>
-
water: 14 &micro;l<br />
+
<div style="margin-left: 40px;"><small>water: 14
 +
&micro;l<br />
BSA: 0.5 &micro;l NEB iGEM stock<br />
BSA: 0.5 &micro;l NEB iGEM stock<br />
buffer: 3 &micro;l buffer 3 NEB iGEM stock<br />
buffer: 3 &micro;l buffer 3 NEB iGEM stock<br />
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Restriction_enzyme_2 : 1 &micro;l SpeI from NEB KuKlabstock <br />
Restriction_enzyme_2 : 1 &micro;l SpeI from NEB KuKlabstock <br />
-&gt; put in 37&deg;C room for 2h
-&gt; put in 37&deg;C room for 2h
-
-&gt;preparative gel<br />
+
-&gt;preparative gel</small><br />
<br />
<br />
-
<table style="text-align: left; width: 623px; height: 529px;"
+
</div>
 +
<table
 +
style="text-align: left; width: 623px; height: 529px; margin-left: 40px;"
  border="0" cellpadding="0" cellspacing="0">
  border="0" cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
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     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>Agarose gel; Lanes: 1.Gene ruler ladder mix of
+
       <td><small>Agarose gel; Lanes: 1.Gene ruler ladder
 +
mix of
fermentas,
fermentas,
-
2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka</td>
+
2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka</small></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<br />
+
<small><br />
&nbsp;interpretation:
&nbsp;interpretation:
bands had right size but pExStrepDigSplitFoka showed
bands had right size but pExStrepDigSplitFoka showed
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- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br />
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br />
<br />
<br />
-
<ul>
+
</small></div>
-
   <li>Inoculation of - pJS419_StrepDigSplitFoka</li>
+
<ul style="margin-left: 40px;">
 +
   <small></small><li>Inoculation of -
 +
pJS419_StrepDigSplitFoka</li>
 +
  <small></small>
</ul>
</ul>
-
- pJS419_HisDigSplitFoka<br />
+
<div style="margin-left: 40px;"><small>-
 +
pJS419_HisDigSplitFoka<br />
- pEx_HisFluA<br />
- pEx_HisFluA<br />
- pEx_HisDigMiddleLiFoka
- pEx_HisDigMiddleLiFoka
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and
   <li>digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and
pMA_scFa-anti_NIP for recloning
pMA_scFa-anti_NIP for recloning
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   </li>
   </li>
</ul>
</ul>
-
<h3>02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell,
+
<h3 style="margin-left: 40px;">02.10.09 Laura, Manu,
 +
Anika, Julia, Hannes, Gerrit, Isabell,
Sarah, Caro, Timo, Christoph, Max</h3>
Sarah, Caro, Timo, Christoph, Max</h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>phage display
   <li>phage display
- OD600 of the pre-culture should be 0,2 </li>
- OD600 of the pre-culture should be 0,2 </li>
</ul>
</ul>
-
- 37&deg;C shaker<br />
+
<div style="margin-left: 40px;"><small>-
 +
37&deg;C shaker<br />
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br />
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br />
- Apportion culture into 10x50 ml Falkon tubes<br />
- Apportion culture into 10x50 ml Falkon tubes<br />
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- Measure OD600 (1:100 dilution), OD should be 0,4<br />
- Measure OD600 (1:100 dilution), OD should be 0,4<br />
- Make aliquots &agrave; 80 &micro;l, souse with N2<br />
- Make aliquots &agrave; 80 &micro;l, souse with N2<br />
-
- Store at -80&deg;C<br />
+
- Store at -80&deg;C</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>glycerol stock of pEx_HisFluASplitFoki in BL21de3
   <li>glycerol stock of pEx_HisFluASplitFoki in BL21de3
   </li>
   </li>
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Digestion:</li>
Digestion:</li>
</ul>
</ul>
-
- digested with xba and
+
<div style="margin-left: 40px;"><small>- digested
 +
with xba and
spe --&gt; GelBilder
spe --&gt; GelBilder
-
<br />
+
</small><br />
-
<div style="margin-left: 40px;"><br />
+
</div>
</div>
-
<ul>
+
<div style="margin-left: 80px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Gelextraction
   <li>&nbsp;Gelextraction
Min Elute Gel Extraction Kit</li>
Min Elute Gel Extraction Kit</li>
</ul>
</ul>
-
- mesure mass: StrepFokA =
+
<div style="margin-left: 40px;"><small>- mesure
 +
mass: StrepFokA =
50 mg<be>
50 mg<be>
- JS418= 90mg</be><br />
- JS418= 90mg</be><br />
<be>-JS419=80mg</be><br />
<be>-JS419=80mg</be><br />
<be>- HisFokA = 250mg</be><br />
<be>- HisFokA = 250mg</be><br />
-
<be style="color: rgb(255, 0, 0);">Nandropdata</be><br />
+
<be style="color: rgb(255, 0, 0);">Nandropdata</be></small><br />
-
<div style="margin-left: 40px;"><be></be></div>
+
</div>
-
<be><br />
+
<div style="margin-left: 80px;">
-
</be>
+
<be></be></div>
-
<ul>
+
<div style="margin-left: 40px;"><be></be><br />
 +
<be></be></div>
 +
<ul style="margin-left: 40px;">
   <li><be>Ligation
   <li><be>Ligation
- with Quickligase
- with Quickligase
JS119-StrepFokA</be></li>
JS119-StrepFokA</be></li>
</ul>
</ul>
-
<be>JS118-Strep</be><br />
+
<div style="margin-left: 40px;"><small><be>JS118-Strep</be><br />
<be>JS118-HisFokA</be><br />
<be>JS118-HisFokA</be><br />
<be>JS19-HisFokA</be><br />
<be>JS19-HisFokA</be><br />
<be>- 15 min at 25&deg;C
<be>- 15 min at 25&deg;C
-
</be><br />
+
</be></small><br />
-
<div style="margin-left: 40px;"><be></be></div>
+
</div>
-
<ul>
+
<div style="margin-left: 80px;">
 +
<be></be></div>
 +
<ul style="margin-left: 40px;">
   <li><be>Transformation
   <li><be>Transformation
- 2YT-Medium 950 &micro;L to 50&micro;l cells and DNA </be></li>
- 2YT-Medium 950 &micro;L to 50&micro;l cells and DNA </be></li>
</ul>
</ul>
-
<be>BL21-JS190</be><br />
+
<div style="margin-left: 40px;"><small><be>BL21-JS190</be><br />
<be>BL21-JS119StrepFokA</be><br />
<be>BL21-JS119StrepFokA</be><br />
<be>BL21-JS118Strep</be><br />
<be>BL21-JS118Strep</be><br />
<be>BL21-JS118HisFokA</be><br />
<be>BL21-JS118HisFokA</be><br />
<be>BL21-JS19HisFokA
<be>BL21-JS19HisFokA
-
- Plated on KM plats</be><br />
+
- Plated on KM plats</be></small><br />
-
<div style="margin-left: 40px;"><be></be></div>
+
</div>
-
<be><br />
+
<div style="margin-left: 80px;">
-
</be>
+
<be></be></div>
-
<ul>
+
<div style="margin-left: 40px;"><be></be><br />
 +
<be></be></div>
 +
<ul style="margin-left: 40px;">
   <li><be>&nbsp;Preparation of overnight cultures 5 ml
   <li><be>&nbsp;Preparation of overnight cultures 5 ml
LB medium each
LB medium each
- pBad+Kan</be></li>
- pBad+Kan</be></li>
</ul>
</ul>
-
<div style="margin-left: 40px;"><be>-pET39B(+)</be><br />
+
<div style="margin-left: 80px;"><small><be>-pET39B(+)</be></small><br />
<be></be></div>
<be></be></div>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li><be>Amp</be></li>
   <li><be>Amp</be></li>
</ul>
</ul>
-
<div style="margin-left: 40px;"><be>--&gt;
+
<div style="margin-left: 80px;"><small><be>--&gt;
Stored in 37&deg;C room
Stored in 37&deg;C room
-
</be><br />
+
</be></small><br />
<be></be></div>
<be></be></div>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li><be>Digest:
   <li><be>Digest:
- pMASplitFoka</be></li>
- pMASplitFoka</be></li>
</ul>
</ul>
-
<be>- pMAFoka</be><br />
+
<div style="margin-left: 40px;"><small><be>-
 +
pMAFoka</be><br />
<be>- pMAFoki</be><br />
<be>- pMAFoki</be><br />
<be>- pMASplitFoki</be><br />
<be>- pMASplitFoki</be><br />
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<be>- pMAShortli</be><br />
<be>- pMAShortli</be><br />
<be>- pMAMiddleli</be><br />
<be>- pMAMiddleli</be><br />
-
<be>- pMALongli</be><br />
+
<be>- pMALongli</be></small><br />
-
<be>- pExStrepFluA</be><br />
+
<small><be>- pExStrepFluA</be><br />
-
<div style="margin-left: 40px;"><be></be></div>
+
</small></div>
-
<be><br />
+
<div style="margin-left: 80px;">
-
</be>
+
<small><be></be></small></div>
-
<ul>
+
<div style="margin-left: 40px;"><be></be><br />
 +
<be></be></div>
 +
<ul style="margin-left: 40px;">
   <li><be>preparative gel
   <li><be>preparative gel
[[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel;
[[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel;
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   <li><be>digest and ligation into new pMA:</be></li>
   <li><be>digest and ligation into new pMA:</be></li>
</ul>
</ul>
-
<be>-pMASplitFokA
+
<div style="margin-left: 40px;"><small><be>-pMASplitFokA
clone1 19.08.09</be><br />
clone1 19.08.09</be><br />
<be>-pMAFokA clone2 05.08.09</be><br />
<be>-pMAFokA clone2 05.08.09</be><br />
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<be>-pMAlongLiFoki clone1 10.09.09</be><br />
<be>-pMAlongLiFoki clone1 10.09.09</be><br />
<be>digestion with EcoRI and SpeI (vector and insert)
<be>digestion with EcoRI and SpeI (vector and insert)
-
</be><br />
+
</be></small><br />
-
<div style="margin-left: 40px;"><be></be></div>
+
</div>
-
<be><br />
+
<div style="margin-left: 80px;">
-
</be>
+
<be></be></div>
-
<ul>
+
<div style="margin-left: 40px;"><be></be><br />
 +
<be></be></div>
 +
<ul style="margin-left: 40px;">
   <li><be>&nbsp;Transformation of:</be></li>
   <li><be>&nbsp;Transformation of:</be></li>
</ul>
</ul>
-
<be>-pMAFokA clone2
+
<div style="margin-left: 40px;"><small><be>-pMAFokA
 +
clone2
05.08.09</be><br />
05.08.09</be><br />
<be> -pMAFoki clone2 05.08.09</be><br />
<be> -pMAFoki clone2 05.08.09</be><br />
-
<be></be>
+
<be></be><be>-pMASplitFoki
-
<be>-pMASplitFoki
+
clone2 06.08.09</be></small><br />
-
clone2 06.08.09</be><br />
+
<be></be><be></be><br />
-
<be></be>
+
<be></be></div>
-
<be><br />
+
<ul style="margin-left: 40px;">
-
</be>
+
-
<ul>
+
   <li><be>plasmidprep of:</be></li>
   <li><be>plasmidprep of:</be></li>
</ul>
</ul>
-
<be>1.
+
<div style="margin-left: 40px;"><small><be>1.
pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be><br />
pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be><br />
<be>2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br />
<be>2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br />
Line 457: Line 490:
<be>11. pEX_DsbA+His+Dig+Split+FokA Clone2 --&gt; seq
<be>11. pEX_DsbA+His+Dig+Split+FokA Clone2 --&gt; seq
02.10.09 Gerrit4</be><br />
02.10.09 Gerrit4</be><br />
-
<be>12. pEX_DsbA+His+Dig+Split+FokA Clone3</be><br />
+
<be>12. pEX_DsbA+His+Dig+Split+FokA Clone3</be></small><br />
-
<be></be>
+
<be></be><be></be><br />
<be></be><br />
<be></be><br />
-
<be></be><br />
+
<be></be></div>
-
<be></be>
+
<ul style="margin-left: 40px;">
-
<ul>
+
   <li><be>testdigest of:
   <li><be>testdigest of:
V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be></li>
V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be></li>
</ul>
</ul>
-
<be>V2.
+
<div style="margin-left: 40px;"><small><be>V2.
pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br />
pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be><br />
<be>V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be><br />
<be>V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be><br />
Line 485: Line 517:
<be>V14. pEX_His+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>with NcoI-HF and XbaI 1h at 37&deg;C</be><br />
-
<be> --&gt; geldbild
+
</small><be><small> --&gt; geldbild</small>
-
</be>
+
</be></div>
-
<h3>03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph </h3>
+
<h3 style="margin-left: 40px;">03.10.09 Manu, Hannes,
-
<br />
+
Gerrit, Caro, Timo, Christoph </h3>
-
<ul>
+
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>plasmid prep of pET and pBad and Glytsocks(Xbl)
   <li>plasmid prep of pET and pBad and Glytsocks(Xbl)
   </li>
   </li>
Line 502: Line 536:
   </li>
   </li>
</ul>
</ul>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)</li>
   <li>digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br />
pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br />
- pEx-Strep-Dig-ShortLinker-Fok(inactive)<br />
- pEx-Strep-Dig-ShortLinker-Fok(inactive)<br />
Line 520: Line 554:
- pMA<br />
- pMA<br />
with NgoMIV and SpeI
with NgoMIV and SpeI
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>digest of
   <li>digest of
- pMA with XbaI and AgeI
- pMA with XbaI and AgeI
   </li>
   </li>
</ul>
</ul>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>Ligation of: - Strep-Dig-LongLinker-Fok(inactive)</li>
   <li>Ligation of: - Strep-Dig-LongLinker-Fok(inactive)</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
Strep-Dig-MiddleLinker-Fok(inactive)<br />
Strep-Dig-MiddleLinker-Fok(inactive)<br />
- trep-Dig-ShortLinker-Fok(inactive)<br />
- trep-Dig-ShortLinker-Fok(inactive)<br />
Line 547: Line 582:
- trep-Dig-ShortLinker-Fok(inactive)<br />
- trep-Dig-ShortLinker-Fok(inactive)<br />
into new pEX
into new pEX
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>inoculation of:
   <li>inoculation of:
-pMASplitFokA clone1 19.08.09</li>
-pMASplitFokA clone1 19.08.09</li>
</ul>
</ul>
-
-pMAFokA clone2 05.08.09
+
<div style="margin-left: 40px;"><small>-pMAFokA
 +
clone2 05.08.09
(new)<br />
(new)<br />
-pMAFoki clone2 05.08.09 (new)<br />
-pMAFoki clone2 05.08.09 (new)<br />
Line 564: Line 601:
-pMAFokA clone2 05.08.09 (old)<br />
-pMAFokA clone2 05.08.09 (old)<br />
-pMAFoki clone2 05.08.09 (old)<br />
-pMAFoki clone2 05.08.09 (old)<br />
-
-pMASplitFoki clone2 06.08.09 (old)<br />
+
-pMASplitFoki clone2 06.08.09 (old)</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>inoculation and plasmid preperation:
   <li>inoculation and plasmid preperation:
-pEX_strepFluA
-pEX_strepFluA
Line 578: Line 616:
   </li>
   </li>
</ul>
</ul>
-
<h3>Plan for 04.10.09</h3>
+
<h3 style="margin-left: 40px;">Plan for 04.10.09</h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>inoculation of
   <li>inoculation of
- pMA-Strep-Dig-LongLinker-Fok(inactive)</li>
- pMA-Strep-Dig-LongLinker-Fok(inactive)</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br />
pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)<br />
Line 597: Line 636:
- pMA-His-FluA-SplitLinker-Fok(inactive)<br />
- pMA-His-FluA-SplitLinker-Fok(inactive)<br />
into LB-Amp
into LB-Amp
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>digestion of pEx-Strep-Flua with AgeI and PstI
   <li>digestion of pEx-Strep-Flua with AgeI and PstI
   </li>
   </li>
Line 609: Line 649:
-pMASplitFokA </li>
-pMASplitFokA </li>
</ul>
</ul>
-
-pMAFokA <br />
+
<div style="margin-left: 40px;"><small>-pMAFokA <br />
-pMAFoki <br />
-pMAFoki <br />
-pMASplitFoki <br />
-pMASplitFoki <br />
Line 619: Line 659:
-pMAFokA (old)<br />
-pMAFokA (old)<br />
-pMAFoki (old)<br />
-pMAFoki (old)<br />
-
-pMASplitFoki (old)<br />
+
-pMASplitFoki (old)</small><br />
-
<h3>04.10.09, Laura, Anika, Max</h3>
+
</div>
-
<br />
+
<h3 style="margin-left: 40px;">04.10.09, Laura, Anika, Max</h3>
-
<ul>
+
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Plasmid preparation of -pMASplitFokA </li>
   <li>&nbsp;Plasmid preparation of -pMASplitFokA </li>
</ul>
</ul>
-
-pMAFokA <br />
+
<div style="margin-left: 40px;"><small>-pMAFokA <br />
-pMAFoki <br />
-pMAFoki <br />
-pMASplitFoki <br />
-pMASplitFoki <br />
Line 638: Line 680:
-pMASplitFoki (old)<br />
-pMASplitFoki (old)<br />
clone 1 and 2,respectively - results see notebook
clone 1 and 2,respectively - results see notebook
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Glycerolstock of </li>
   <li>&nbsp;Glycerolstock of </li>
</ul>
</ul>
-
-pMASplitFokA <br />
+
<div style="margin-left: 40px;"><small>-pMASplitFokA
 +
<br />
-pMAFokA <br />
-pMAFokA <br />
-pMAFoki <br />
-pMAFoki <br />
Line 654: Line 698:
-pMAFokA (old)<br />
-pMAFokA (old)<br />
-pMAFoki (old)<br />
-pMAFoki (old)<br />
-
-pMASplitFoki (old)<br />
+
-pMASplitFoki (old)</small><br />
-
clone 3
+
<small>clone 3
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Pellets of</li>
   <li>&nbsp;Pellets of</li>
</ul>
</ul>
-
-pMASplitFokA <br />
+
<div style="margin-left: 40px;"><small>-pMASplitFokA
 +
<br />
-pMAFokA <br />
-pMAFokA <br />
-pMAFoki <br />
-pMAFoki <br />
Line 675: Line 721:
clone 1 and 2,respectively
clone 1 and 2,respectively
- stored in Pelletbox, -20&deg;C
- stored in Pelletbox, -20&deg;C
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Digest of 1. pEXHisDigMiddleLFoki
   <li>Digest of 1. pEXHisDigMiddleLFoki
2. pMaScFaantiNIP
2. pMaScFaantiNIP
Line 682: Line 729:
4. pMAShortLFoka</li>
4. pMAShortLFoka</li>
</ul>
</ul>
-
5. pMAShortLFoki<br />
+
<div style="margin-left: 40px;"><small>5.
 +
pMAShortLFoki<br />
6. pMAMiddleLFoka<br />
6. pMAMiddleLFoka<br />
7. pMAMiddleLFoki<br />
7. pMAMiddleLFoki<br />
8. pMALongLFoka<br />
8. pMALongLFoka<br />
9. pMALongLFoki<br />
9. pMALongLFoki<br />
-
10.pEXStrepFlua<br />
+
10.pEXStrepFlua</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Preparative Gel of Digest</li>
   <li>Preparative Gel of Digest</li>
</ul>
</ul>
-
<table style="text-align: left; width: 750px;" border="0"
+
<table style="text-align: center; width: 750px; margin-left: 40px;"
-
cellpadding="0" cellspacing="0">
+
border="0" cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
-
       <td><img style="width: 759px; height: 575px;"
+
       <td align="center"><img style="width: 380px; height: 288px;"
  alt=""
  alt=""
  src="https://static.igem.org/mediawiki/2009/d/df/Freiburg09_021009_umklonierung.JPG" /></td>
  src="https://static.igem.org/mediawiki/2009/d/df/Freiburg09_021009_umklonierung.JPG" /></td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki,
+
       <td><small></small><small>Agarose gel;
 +
Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki,
4.pMASplitFoki,
4.pMASplitFoki,
5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli,
5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli,
-
9.pMALongli, 10.pExStrepFluA</td>
+
9.pMALongli, 10.pExStrepFluA</small></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
 +
<div style="margin-left: 40px;"><br />
<br />
<br />
-
<br />
+
</div>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>Gelextraction</li>
   <li>Gelextraction</li>
   <li>Ligation of</li>
   <li>Ligation of</li>
</ul>
</ul>
 +
<div style="margin-left: 40px;"><small>
1.pMAHisDigMiddleFoki<br />
1.pMAHisDigMiddleFoki<br />
2.pMACAT<br />
2.pMACAT<br />
Line 728: Line 780:
Insert, 2&micro;l Quick Ligase Buffer, 1 &micro;l Quick Ligase,
Insert, 2&micro;l Quick Ligase Buffer, 1 &micro;l Quick Ligase,
15 min, Room Temp.<br />
15 min, Room Temp.<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Dephosphorylation of pBAD Vector: </li>
   <li>Dephosphorylation of pBAD Vector: </li>
</ul>
</ul>
-
Approach: 2&micro;l
+
<div style="margin-left: 40px;"><small>Approach:
 +
2&micro;l
Eluat, 3,1 &micro;l Fast Ap Buffer 10x, 1&micro;l Fast Ap, 10
Eluat, 3,1 &micro;l Fast Ap Buffer 10x, 1&micro;l Fast Ap, 10
-
min. 37&deg;C, 5 min. 75&deg;C<br />
+
min. 37&deg;C, 5 min. 75&deg;C</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Transformation of</li>
   <li>Transformation of</li>
</ul>
</ul>
-
1.pMAHisDigMiddleFoki (RV)<br />
+
<div style="margin-left: 40px;"><small>1.pMAHisDigMiddleFoki
 +
(RV)<br />
2.pMACAT(RV)<br />
2.pMACAT(RV)<br />
3.pEXStrepFluAShortLFokA(RV)<br />
3.pEXStrepFluAShortLFokA(RV)<br />
Line 750: Line 806:
10.pJS 419StrepDigSplitFokA(XBL)<br />
10.pJS 419StrepDigSplitFokA(XBL)<br />
11.pJS 419HisDigSplitFokA(XBL)<br />
11.pJS 419HisDigSplitFokA(XBL)<br />
-
12.pJS 418HisDigSplitFokA(XBL)<br />
+
12.pJS 418HisDigSplitFokA(XBL)</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>test digest of plasmid preps from today
   <li>test digest of plasmid preps from today
-&gt; run on a gel with digest pET39, xbaI, ecoRI from Tobi
-&gt; run on a gel with digest pET39, xbaI, ecoRI from Tobi
Line 767: Line 824:
   </li>
   </li>
</ul>
</ul>
-
<h3>05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes,
+
<h3 style="margin-left: 40px;">05.10.09, Laura, Manu,
 +
Julia, Christophe, Caro, Hannes,
Gerrit, Anika</h3>
Gerrit, Anika</h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;3l LB and 2L LB Agar
   <li>&nbsp;3l LB and 2L LB Agar
   </li>
   </li>
Line 783: Line 842:
   <li>1) Gelextraction</li>
   <li>1) Gelextraction</li>
</ul>
</ul>
-
--&gt; Gelbild <br />
+
<div style="margin-left: 40px;"><small>--&gt;
 +
Gelbild <br />
1) Vector: pEXHisFluSplitFoki<br />
1) Vector: pEXHisFluSplitFoki<br />
2)Insert: RBSStrepDigSplitFokA<br />
2)Insert: RBSStrepDigSplitFokA<br />
-
3)PET39b+<br />
+
3)PET39b+</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>2)Ligation</li>
   <li>2)Ligation</li>
</ul>
</ul>
-
-10ul 2 fold buffer<br />
+
<div style="margin-left: 40px;"><small>-10ul 2 fold
 +
buffer<br />
- 6 &micro;l Insert<br />
- 6 &micro;l Insert<br />
-3&micro;l Vector<br />
-3&micro;l Vector<br />
-1&micro;l Quick Ligase<br />
-1&micro;l Quick Ligase<br />
--&gt; 15 min at RT<br />
--&gt; 15 min at RT<br />
-
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA<br />
+
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>3)Transformation</li>
   <li>3)Transformation</li>
</ul>
</ul>
-
-5 &micro;l DNA of
+
<div style="margin-left: 40px;"><small>-5
 +
&micro;l DNA of
Ligation + BL21d3 and XBL
Ligation + BL21d3 and XBL
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>'''Mini prep with with Qiagen Spin Miniprep Kit of'''
   <li>'''Mini prep with with Qiagen Spin Miniprep Kit of'''
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li>
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br />
pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br />
Line 821: Line 886:
- pMA-His-FluA-SplitLinker-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 />
+
</small><br />
-
<ul>
+
</div>
-
   <li>'''Made Glycerolstocks of'''
+
<ul style="margin-left: 40px;">
 +
   <li>Made Glycerolstocks of
   </li>
   </li>
   <li>700&micro;l Cellsuspension+ 300&micro;l Glycerol
   <li>700&micro;l Cellsuspension+ 300&micro;l Glycerol
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li>
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br />
pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br />
Line 841: Line 907:
- pMA-His-FluA-SplitLinker-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 />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>'''Test Digestion of'''
   <li>'''Test Digestion of'''
   </li>
   </li>
Line 850: Line 917:
- pMA-Strep-Dig-LongLinker-Fok(inactive)3</li>
- pMA-Strep-Dig-LongLinker-Fok(inactive)3</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
pMA-Strep-Dig-MiddleLinker-Fok(inactive)1<br />
pMA-Strep-Dig-MiddleLinker-Fok(inactive)1<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1<br />
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1<br />
Line 862: Line 929:
- pMA-His-FluA-ShortLinker-Fok(inactive)1<br />
- pMA-His-FluA-ShortLinker-Fok(inactive)1<br />
- pMA-His-FluA-SplitLinker-Fok(inactive)2<br />
- pMA-His-FluA-SplitLinker-Fok(inactive)2<br />
-
- pMA-Dbsa 2<br />
+
- pMA-Dbsa 2</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Transformation of</li>
   <li>&nbsp;Transformation of</li>
</ul>
</ul>
-
- XBL
+
<div style="margin-left: 40px;"><small>- XBL
pEXHisFluASplitFokIRBSStrepDigSplitFokA<br />
pEXHisFluASplitFokIRBSStrepDigSplitFokA<br />
-
- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA<br />
+
- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l
   <li>Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l
flasks in 600ml DYT medium each
flasks in 600ml DYT medium each
- induced with 0,7mM IPTG and took samples T0-T5</li>
- induced with 0,7mM IPTG and took samples T0-T5</li>
</ul>
</ul>
-
- centrifuged in buckets
+
<div style="margin-left: 40px;"><small>- centrifuged
 +
in buckets
17', 4000rpm, 4&deg;C<br />
17', 4000rpm, 4&deg;C<br />
- eluted in 20 ml TES in each bucket<br />
- eluted in 20 ml TES in each bucket<br />
....
....
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>PET 39 b+ ssDNA "PCR" with new template from digestion of
   <li>PET 39 b+ ssDNA "PCR" with new template from digestion of
yesterday -&gt; very few product, as well as non specific ones
yesterday -&gt; very few product, as well as non specific ones
Line 889: Line 960:
   </li>
   </li>
</ul>
</ul>
-
<h3>06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes,
+
<h3 style="margin-left: 40px;">06.10.09, Laura, Manu,
 +
Julia, Christophe, Caro, Hannes,
Gerrit, Anika</h3>
Gerrit, Anika</h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>digest
   <li>digest
- pBAD vector </li>
- pBAD vector </li>
</ul>
</ul>
-
DNA: 16.8&micro;l<br />
+
<div style="margin-left: 40px;"><small>DNA:
 +
16.8&micro;l<br />
enzyme: 1&micro;l AgeI<br />
enzyme: 1&micro;l AgeI<br />
buffer: 2&micro;l buffer 1 <br />
buffer: 2&micro;l buffer 1 <br />
Line 913: Line 987:
enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/<br />
enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/<br />
buffer: 3&micro;l buffer 3 <br />
buffer: 3&micro;l buffer 3 <br />
-
BSA (10X): 3&micro;l <br />
+
BSA (10X): 3&micro;l </small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA:
   <li>testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA:
5&micro;l</li>
5&micro;l</li>
</ul>
</ul>
-
enzyme: 1&micro;l PstI
+
<div style="margin-left: 40px;"><small>enzyme:
 +
1&micro;l PstI
and 1.5 &micro;l Xba/<br />
and 1.5 &micro;l Xba/<br />
buffer: 1&micro;l buffer 3 <br />
buffer: 1&micro;l buffer 3 <br />
-
BSA (10X): 1&micro;l <br />
+
BSA (10X): 1&micro;l </small><br />
<br />
<br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>preparative gels of digests
   <li>preparative gels of digests
   </li>
   </li>
</ul>
</ul>
-
<div style="margin-left: 40px;"></div>
+
<div style="margin-left: 80px;"></div>
-
<table style="text-align: left; width: 750px;" border="1"
+
<table style="text-align: left; width: 750px; margin-left: 40px;"
-
cellpadding="0" cellspacing="1">
+
border="1" cellpadding="0" cellspacing="1">
   <tbody>
   <tbody>
     <tr>
     <tr>
Line 942: Line 1,019:
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>preparative agarose gel, lanes: 1. Genruler DNA ladder
+
       <td><small>preparative agarose gel, lanes: 1.
-
mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418</td>
+
Genruler DNA ladder
-
       <td>preparative agarose gel, lanes: 1. Genruler DNA ladder
+
mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418</small></td>
-
mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka</td>
+
       <td><small>preparative agarose gel, lanes: 1.
 +
Genruler DNA ladder
 +
mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka</small></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
 +
<div style="margin-left: 40px;"><small><br />
interpretation:
interpretation:
pExStrepDigSplitFoka seemed
pExStrepDigSplitFoka seemed
Line 954: Line 1,034:
size even if the concentration of the PCR pBAD insert seems to be very
size even if the concentration of the PCR pBAD insert seems to be very
low
low
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>testdigest of pMAdsba
   <li>testdigest of pMAdsba
DNA: 5&micro;l</li>
DNA: 5&micro;l</li>
</ul>
</ul>
-
enzyme: 1&micro;l PstI
+
<div style="margin-left: 40px;"><small>enzyme:
 +
1&micro;l PstI
and 1.5 &micro;l Xba/<br />
and 1.5 &micro;l Xba/<br />
buffer: 1&micro;l buffer 3 <br />
buffer: 1&micro;l buffer 3 <br />
-
BSA (10X): 1&micro;l <br />
+
BSA (10X): 1&micro;l </small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>His-tag purification of HisDigSplitFoka (periplasm export)
   <li>His-tag purification of HisDigSplitFoka (periplasm export)
with Ni-NTA
with Ni-NTA
Line 972: Line 1,055:
   </li>
   </li>
</ul>
</ul>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>Ligation
   <li>Ligation
- Dephphorylation of pBAD Gelex</li>
- Dephphorylation of pBAD Gelex</li>
</ul>
</ul>
-
- 1&micro;l Fast AP<br />
+
<div style="margin-left: 40px;"><small>-
 +
1&micro;l Fast AP<br />
- 5.5&micro;l fast AP buffer<br />
- 5.5&micro;l fast AP buffer<br />
-1.5&micro;l water<br />
-1.5&micro;l water<br />
Line 985: Line 1,069:
-1&micro;l Quickligase<br />
-1&micro;l Quickligase<br />
-10&micro;l buffer<br />
-10&micro;l buffer<br />
-
--&gt; pBAD <br />
+
--&gt; pBAD </small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Insert (Dummy)</li>
   <li>&nbsp;Insert (Dummy)</li>
</ul>
</ul>
-
--&gt;
+
<div style="margin-left: 40px;"><small>--&gt;
pJS419+HisDigSplitFoka
pJS419+HisDigSplitFoka
--&gt;pJ418+HisDigSplitFoka
--&gt;pJ418+HisDigSplitFoka
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Transformation
   <li>&nbsp;Transformation
--&gt; pBAD </li>
--&gt; pBAD </li>
   <li>&nbsp;Insert (Dummy)</li>
   <li>&nbsp;Insert (Dummy)</li>
</ul>
</ul>
-
--&gt;
+
<div style="margin-left: 40px;"><small>--&gt;
pJS419+HisDigSplitFoka
pJS419+HisDigSplitFoka
--&gt;pJ418+HisDigSplitFoka
--&gt;pJ418+HisDigSplitFoka
- in XLblue<br />
- in XLblue<br />
-pBAD was plated on AMP plates<br />
-pBAD was plated on AMP plates<br />
-
- Both pJS... were plated on CM plates<br />
+
- Both pJS... were plated on CM plates</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li><big>Ligation and Transformation of</big></li>
   <li><big>Ligation and Transformation of</big></li>
</ul>
</ul>
-
&nbsp;Ligation:per
+
<div style="margin-left: 40px;">&nbsp;<small>Ligation:per
sample 8&micro;l H2O; 3&micro;l vector;
sample 8&micro;l H2O; 3&micro;l vector;
6&micro;l insert; 2&micro;l Quick Ligase buffer; 1&micro;l
6&micro;l insert; 2&micro;l Quick Ligase buffer; 1&micro;l
Line 1,031: Line 1,118:
-pEX Strep FluA ML Foki<br />
-pEX Strep FluA ML Foki<br />
-pEX Strep FluA ML Foka<br />
-pEX Strep FluA ML Foka<br />
-
-pEX Strep FluA LL Foka<br />
+
-pEX Strep FluA LL Foka</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Sequencing:
   <li>Sequencing:
27&micro;l H2O; 3&micro;l DNA
27&micro;l H2O; 3&micro;l DNA
-pMA Dbsa clone 2</li>
-pMA Dbsa clone 2</li>
</ul>
</ul>
-
-pMA HisDigSplitFokA clone
+
<div style="margin-left: 40px;"><small>-pMA
 +
HisDigSplitFokA clone
2<br />
2<br />
-pMA HisFluASL Foki clone 1<br />
-pMA HisFluASL Foki clone 1<br />
Line 1,050: Line 1,139:
-pMA StrepDigSplitFoki clone 3<br />
-pMA StrepDigSplitFoki clone 3<br />
-pMA HisDigSplitFoki clone 1<br />
-pMA HisDigSplitFoki clone 1<br />
-
-pMA HisFluaLLFoki clone 3<br />
+
-pMA HisFluaLLFoki clone 3</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Inoculation
   <li>Inoculation
of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37&deg;c room
of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37&deg;c room
Line 1,072: Line 1,162:
   </li>
   </li>
</ul>
</ul>
-
<h3>07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika</h3>
+
<small>
-
<br />
+
FokI wildtype cutting<br>
-
<ul>
+
 
 +
Hybridize M13 ssDNA with Fok control oligos 2 and 3
 +
<table border="1">
 +
<tr>
 +
<td align="center">  Volume  </td>
 +
<td>Reagent</td>
 +
</tr>
 +
<tr>
 +
<td align="center">10 µl</td>
 +
<td>M13 ssDNA (c = 127 ng/µl, 17.08.09, sample #5.2)</td>
 +
</tr>
 +
<tr>
 +
<td align="center">5 µl</td>
 +
<td>MgCl2 (5 mM)</td>
 +
</tr>
 +
<tr>
 +
<td align="center">5 µl</td>
 +
<td>Tris-HCl pH 8 (100 mM)</td>
 +
</tr>
 +
<tr>
 +
<td align="center">2 µl</td>
 +
<td>Fok control 2 (10 ng/µl; 1 µM)</td>
 +
</tr>
 +
<tr>
 +
<td align="center">2 µl</td>
 +
<td>Fok control 3 (10 ng/µl; 1 µM)</td>
 +
</tr>
 +
<tr>
 +
<td align="center">30 µl</td>
 +
<td></td>
 +
</tr>
 +
</table><br>
 +
The hybridization was also done with just Fok control 2 and with no oligos at all.<br>
 +
Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).<br>
 +
 
 +
10 µl of the hybridized DNA were used for a cutting experiment with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI (wildtype Fok from NEB). The digest was incubated for 30 minutes at 37°C.<br>
 +
 
 +
 
 +
<table>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2009/5/5a/Freiburg09_061009_fokI_cutting2_crop.JPG" name="Agarose gel of FokI cutting M13 ssDNA" width="188" height="230" /><br>
 +
Agarose gel of FokI cutting M13 ssDNA
 +
</td>
 +
 
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2009/4/4f/Freiburg09_061009_fokI_cutting3_crop.jpg" name="Agarose gel of FokI cutting M13 ssDNA"width="188" height="230" /><br>
 +
Agarose gel of FokI cutting M13 ssDNA
 +
</td>
 +
</tr>
 +
</table>
 +
 
 +
Lane 1: GeneRuler DNA Ladder Mix (Fermentas, 6 µl); <br>
 +
Lane 2: Untreated M13 ssDNA (250 ng); <br>
 +
Lane 3: Digest of M13 ssDNA with FokI and without oligo; <br>
 +
Lane 4: Digest of M13 ssDNA with FokI and with one oligo; <br>
 +
Lane 5: Digest of M13 ssDNA with FokI and two oligos.</small><br><br>
 +
 
 +
 
 +
 
 +
 
 +
<h3 style="margin-left: 40px;">07.10.09 Laura,Christoph,
 +
Hannes, Timo, Julia, Caro, Anika</h3>
 +
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>SDS gel of protein purification of HisDigSplitFoka
   <li>SDS gel of protein purification of HisDigSplitFoka
(periplasm)
(periplasm)
Line 1,086: Line 1,241:
   </li>
   </li>
</ul>
</ul>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>&nbsp;Inoculation 4 Clones respectively:</li>
   <li>&nbsp;Inoculation 4 Clones respectively:</li>
</ul>
</ul>
-
-
+
<div style="margin-left: 40px;"><small>-
pMA-His-Dig-MiddleLinker-Foki in XLBlue<br />
pMA-His-Dig-MiddleLinker-Foki in XLBlue<br />
- pJS419-HIs-Dig-Split-Foka- in XLBlue<br />
- pJS419-HIs-Dig-Split-Foka- in XLBlue<br />
Line 1,100: Line 1,255:
-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 />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Starter culture of pEx-DsbAHisDigSplitFoka in
   <li>&nbsp;Starter culture of pEx-DsbAHisDigSplitFoka in
Bl21de3
Bl21de3
Line 1,108: Line 1,264:
- program name: Assembly</li>
- program name: Assembly</li>
</ul>
</ul>
-
- three different samples:
+
<div style="margin-left: 40px;"><small>- three
 +
different samples:
1. without DMSO, 2. with DMSO, 3. without
1. without DMSO, 2. with DMSO, 3. without
DMSO and with last primers just added after first step
DMSO and with last primers just added after first step
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>preparative gel of the PCR samples
   <li>preparative gel of the PCR samples
-&gt; primer haven't been diluted an probably made all secondary
-&gt; primer haven't been diluted an probably made all secondary
Line 1,125: Line 1,283:
DNA: 5&micro;l</li>
DNA: 5&micro;l</li>
</ul>
</ul>
-
Enzymes: 0.5&micro;l
+
<div style="margin-left: 40px;"><small>Enzymes:
 +
0.5&micro;l
of BamHI and MfeI each<br />
of BamHI and MfeI each<br />
Buffer: 1&micro;l buffer 4<br />
Buffer: 1&micro;l buffer 4<br />
Line 1,131: Line 1,290:
Water: 2&micro;l<br />
Water: 2&micro;l<br />
-&gt; pJS419_HisDigSplitFoka showed bands of the right size and was
-&gt; pJS419_HisDigSplitFoka showed bands of the right size and was
-
sequenced with primer sf_lac P1<br />
+
sequenced with primer sf_lac P1</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>plasmidpreparation of - pEXHisFluASplitFoki</li>
   <li>plasmidpreparation of - pEXHisFluASplitFoki</li>
</ul>
</ul>
-
- pExStrepDigSplitFoka<br />
+
<div style="margin-left: 40px;"><small>-
-
-&gt; low concentrations<br />
+
pExStrepDigSplitFoka<br />
 +
-&gt; low concentrations</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and
   <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and
RBS_StrepDigSplitFoka
RBS_StrepDigSplitFoka
pExHisFluASplitFoki: 15&micro;l</li>
pExHisFluASplitFoki: 15&micro;l</li>
</ul>
</ul>
-
Enzymes: 1&micro;l of
+
<div style="margin-left: 40px;"><small>Enzymes:
 +
1&micro;l of
PstI and 1.5&micro;l SpeI<br />
PstI and 1.5&micro;l SpeI<br />
Buffer: 3&micro;l buffer 2<br />
Buffer: 3&micro;l buffer 2<br />
Line 1,158: Line 1,321:
Buffer: 3&micro;l buffer 3<br />
Buffer: 3&micro;l buffer 3<br />
BSA (100fold): 0.5&micro;l<br />
BSA (100fold): 0.5&micro;l<br />
-
Water: 14&micro;l<br />
+
Water: 14&micro;l</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Gelextraction of digest
   <li>&nbsp;Gelextraction of digest
1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) </li>
1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) </li>
</ul>
</ul>
-
2)Vector:
+
<div style="margin-left: 40px;"><small>2)Vector:
pEX-His-Flua-Split-Foki (digested with spe, pst)<br />
pEX-His-Flua-Split-Foki (digested with spe, pst)<br />
-
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br />
+
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>preparative gels of digests
   <li>preparative gels of digests
   </li>
   </li>
</ul>
</ul>
-
<table border="0">
+
<table style="text-align: left; width: 750px; margin-left: 40px;" border="1"
 +
cellpadding="2" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
-
       <td>[[Image:|none|thumb|preparative agarose gel, lanes: 1.
+
       <td><img style="width: 400px; height: 303px;"
-
Insert2. pEX-Vector, 3. PCR- RBS Product]]
+
alt=""
-
      </td>
+
src="https://static.igem.org/mediawiki/2009/0/0a/Freiburg09_7.10_digestcaro.JPG"></td>
-
       <td>[[Image: |none|thumb|preparative agarose gel, lanes: 1.
+
      <td><img style="width: 400px; height: 303px;"
-
Genruler
+
alt=""
-
DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4.
+
src="https://static.igem.org/mediawiki/2009/f/f3/Freiburg09_7.10_digest2_caro.JPG"></td>
-
pExStrepDigSplitFoka |300x300px]]
+
    </tr>
-
      </td>
+
    <tr>
 +
      <td><small>preparative agarose gel, lanes: 1.
 +
Insert2. pEX-Vector, 3. PCR- RBS Product</small></td>
 +
       <td><small>preparative agarose gel, lanes: 1.
 +
Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4.
 +
pExStrepDigSplitFoka</small></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
RBS PCR Product: m= 90 mg<br />
+
 
-
vector: 0 0 130 mg<br />
+
<div style="margin-left: 40px;"><small>RBS PCR
 +
Product: m= 90 mg<br />
 +
vector: 0 0 130 mg</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Ligation
   <li>&nbsp;Ligation
- 6&micro;l Insert</li>
- 6&micro;l Insert</li>
</ul>
</ul>
-
-3&micro;l Vetor<br />
+
<div style="margin-left: 40px;"><small>-3&micro;l
 +
Vetor<br />
-1&micro;l Quickligase<br />
-1&micro;l Quickligase<br />
-10&micro;l buffer<br />
-10&micro;l buffer<br />
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br />
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Transformation
   <li>Transformation
- All in XL1blue</li>
- All in XL1blue</li>
</ul>
</ul>
-
- Vector:
+
<div style="margin-left: 40px;"><small>- Vector:
pEX-strep-Duig-Split-Foka<br />
pEX-strep-Duig-Split-Foka<br />
-
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br />
+
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Stardet production of Phages baering the phagmid vektors
   <li>Stardet production of Phages baering the phagmid vektors
with
with
Line 1,219: Line 1,396:
   </li>
   </li>
</ul>
</ul>
-
<h3>08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes </h3>
+
<h3 style="margin-left: 40px;">08.10.09 Manu, Christoph,
-
<br />
+
Julia, Caro, Timo, Hannes </h3>
-
<ul>
+
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>Poured SDS-Gels</li>
   <li>Poured SDS-Gels</li>
   <li>Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet
   <li>Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet
Line 1,227: Line 1,406:
OD600=0,4 in 50ml LB+Tet</li>
OD600=0,4 in 50ml LB+Tet</li>
</ul>
</ul>
 +
<div style="margin-left: 40px;"><small>
-Transfomation with 3&micro;l M13 Phage stock (-80&deg;) at
-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 />
10:30Uhr , incubated for 4-5 hours at 37&deg;C in shaker<br />
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4&deg;C<br />
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4&deg;C<br />
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml
-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 />
+
precipitation over night on ice in -4&deg;C room</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Miniprep
   <li>Miniprep
- 2 clones respectively:</li>
- 2 clones respectively:</li>
</ul>
</ul>
-
-A = pEX strep dig split
+
<div style="margin-left: 40px;"><small>-A = pEX
 +
strep dig split
foka
foka
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Finished Phage production(see protocol day 2): We obtained
   <li>Finished Phage production(see protocol day 2): We obtained
approximately 3.8</li>
approximately 3.8</li>
Line 1,248: Line 1,431:
the anti-Flag ELISA</li>
the anti-Flag ELISA</li>
</ul>
</ul>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Anti-Flag ELISA was successfull with a slight but detctable
   <li>Anti-Flag ELISA was successfull with a slight but detctable
signal via
signal via
anti M13 VCS Antibodies (with peroxidase):</li>
anti M13 VCS Antibodies (with peroxidase):</li>
</ul>
</ul>
-
see
+
<div style="margin-left: 40px;"><small>see
405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to
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
positive control (G5-6) and negative control (G3-4 and D3-10) detected
Line 1,260: Line 1,444:
<br />
<br />
<br />
<br />
-
<table id="table_1" border="2" cellpadding="2"
+
</small></div>
-
cellspacing="0">
+
<table style="margin-left: 40px;" id="table_1"
-
   <tbody>
+
border="2" cellpadding="2" cellspacing="0">
 +
   <small></small><tbody>
     <tr>
     <tr>
-
       <td>&nbsp;</td>
+
       <small></small><td><small>&nbsp;</small></td>
-
       <td>1</td>
+
       <small></small><td><small>1</small></td>
-
       <td>2</td>
+
       <small></small><td><small>2</small></td>
-
       <td>3</td>
+
       <small></small><td><small>3</small></td>
-
       <td>4</td>
+
       <small></small><td><small>4</small></td>
-
       <td>5</td>
+
       <small></small><td><small>5</small></td>
-
       <td>6</td>
+
       <small></small><td><small>6</small></td>
-
       <td>7</td>
+
       <small></small><td><small>7</small></td>
-
       <td>8</td>
+
       <small></small><td><small>8</small></td>
-
       <td>9</td>
+
       <small></small><td><small>9</small></td>
-
       <td>10</td>
+
       <small></small><td><small>10</small></td>
-
       <td>11</td>
+
       <small></small><td><small>11</small></td>
-
       <td>12</td>
+
       <small></small><td><small>12</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>A</td>
+
       <small></small><td><small>A</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0420</td>
+
       <small></small><td><small>0.0420</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
       <td>0.0510</td>
+
       <small></small><td><small>0.0510</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>B</td>
+
       <small></small><td><small>B</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0560</td>
+
       <small></small><td><small>0.0560</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>C</td>
+
       <small></small><td><small>C</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0500</td>
+
       <small></small><td><small>0.0500</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0420</td>
+
       <small></small><td><small>0.0420</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>D</td>
+
       <small></small><td><small>D</small></td>
-
       <td>0.0490</td>
+
       <small></small><td><small>0.0490</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.1590</td>
+
       <small></small><td><small>0.1590</small></td>
-
       <td>0.1610</td>
+
       <small></small><td><small>0.1610</small></td>
-
       <td>0.1920</td>
+
       <small></small><td><small>0.1920</small></td>
-
       <td>0.1720</td>
+
       <small></small><td><small>0.1720</small></td>
-
       <td>0.2210</td>
+
       <small></small><td><small>0.2210</small></td>
-
       <td>0.2500</td>
+
       <small></small><td><small>0.2500</small></td>
-
       <td>0.2130</td>
+
       <small></small><td><small>0.2130</small></td>
-
       <td>0.2680</td>
+
       <small></small><td><small>0.2680</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>E</td>
+
       <small></small><td><small>E</small></td>
-
       <td>0.0490</td>
+
       <small></small><td><small>0.0490</small></td>
-
       <td>0.0490</td>
+
       <small></small><td><small>0.0490</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0490</td>
+
       <small></small><td><small>0.0490</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0490</td>
+
       <small></small><td><small>0.0490</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0420</td>
+
       <small></small><td><small>0.0420</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>F</td>
+
       <small></small><td><small>F</small></td>
-
       <td>0.0490</td>
+
       <small></small><td><small>0.0490</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0500</td>
+
       <small></small><td><small>0.0500</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>G</td>
+
       <small></small><td><small>G</small></td>
-
       <td>0.0500</td>
+
       <small></small><td><small>0.0500</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.2530</td>
+
       <small></small><td><small>0.2530</small></td>
-
       <td>0.2510</td>
+
       <small></small><td><small>0.2510</small></td>
-
       <td>3.9180</td>
+
       <small></small><td><small>3.9180</small></td>
-
       <td>3.8800</td>
+
       <small></small><td><small>3.8800</small></td>
-
       <td>0.9560</td>
+
       <small></small><td><small>0.9560</small></td>
-
       <td>0.9380</td>
+
       <small></small><td><small>0.9380</small></td>
-
       <td>1.2420</td>
+
       <small></small><td><small>1.2420</small></td>
-
       <td>1.2360</td>
+
       <small></small><td><small>1.2360</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
    </tr>
+
      <small></small></tr>
-
     <tr>
+
     <small></small><tr>
-
       <td>H</td>
+
       <small></small><td><small>H</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
       <td>0.0430</td>
+
       <small></small><td><small>0.0430</small></td>
-
       <td>0.0510</td>
+
       <small></small><td><small>0.0510</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0480</td>
+
       <small></small><td><small>0.0480</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0470</td>
+
       <small></small><td><small>0.0470</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0450</td>
+
       <small></small><td><small>0.0450</small></td>
-
       <td>0.0440</td>
+
       <small></small><td><small>0.0440</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
       <td>0.0460</td>
+
       <small></small><td><small>0.0460</small></td>
-
    </tr>
+
      <small></small></tr>
 +
    <small></small>
   </tbody>
   </tbody>
</table>
</table>
-
&nbsp; &nbsp;&nbsp;&nbsp;<br />
+
<div style="margin-left: 40px;">&nbsp;
 +
&nbsp;&nbsp;&nbsp;<br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Made new PCR for ssDNA from pET39b+ fragment and
   <li>&nbsp;Made new PCR for ssDNA from pET39b+ fragment and
gained approximately
gained approximately
Line 1,411: Line 1,599:
pExHisFluASplitFoki: 15&micro;l</li>
pExHisFluASplitFoki: 15&micro;l</li>
</ul>
</ul>
-
Enzymes: 1&micro;l of PstI and 1.5&micro;l SpeI<br />
+
<div style="margin-left: 40px;"><small>Enzymes:
 +
1&micro;l of PstI and 1.5&micro;l SpeI<br />
Buffer: 3&micro;l buffer 2<br />
Buffer: 3&micro;l buffer 2<br />
BSA (100fold): 0.5&micro;l<br />
BSA (100fold): 0.5&micro;l<br />
Line 1,426: Line 1,615:
Water: 19&micro;l<br />
Water: 19&micro;l<br />
-&gt;made two digest of PCR construct<br />
-&gt;made two digest of PCR construct<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Gelextraction of digest
   <li>&nbsp;Gelextraction of digest
1) Insert: His-Dig-Split-Foka( digested with xba and pst) </li>
1) Insert: His-Dig-Split-Foka( digested with xba and pst) </li>
</ul>
</ul>
-
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br />
+
<div style="margin-left: 40px;"><small>2)Vector:
 +
pEX-His-Flua-Split-Foki (digested with spe, pst)<br />
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br />
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 />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>preparative gels of digests
   <li>preparative gels of digests
-&gt;see picture</li>
-&gt;see picture</li>
</ul>
</ul>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>ligation of
   <li>ligation of
-pEX-His-Flua-Split-Foki_His-Dig-Split-Foka</li>
-pEX-His-Flua-Split-Foki_His-Dig-Split-Foka</li>
</ul>
</ul>
-
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from gelex)<br />
+
<div style="margin-left: 40px;"><small>-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka
 +
(from gelex)<br />
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR
-
purification)<br />
+
purification)</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>transformation of ligations
   <li>transformation of ligations
   </li>
   </li>
Line 1,459: Line 1,654:
-&gt;interpretation: no band of 921bp was visible</li>
-&gt;interpretation: no band of 921bp was visible</li>
</ul>
</ul>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and
   <li>one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and
preparative agarose gel
preparative agarose gel
-&gt;see picture</li>
-&gt;see picture</li>
</ul>
</ul>
-
-&gt;interpretation: pMAFos and pMASplitFoka showed bands of the
+
<div style="margin-left: 40px;"><small>-&gt;interpretation:
 +
pMAFos and pMASplitFoka showed bands of the
right
right
-
size (230bp and 657bp respectively), of pMADsbA no product was visible<br />
+
size (230bp and 657bp respectively), of pMADsbA no product was visible</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>new one step PCR of pMADsba with newly prepared 1:1000
   <li>new one step PCR of pMADsba with newly prepared 1:1000
dilution
dilution
Line 1,476: Line 1,674:
-&gt;see picture</li>
-&gt;see picture</li>
</ul>
</ul>
-
<h3>09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes,
+
<h3 style="margin-left: 40px;">09.10.09 Manu, Julia, Caro,
 +
Laura, Christopherus, Hannes,
Timo, Max, Anika </h3>
Timo, Max, Anika </h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Phage ss DNA</li>
   <li>Phage ss DNA</li>
</ul>
</ul>
-
-Over night in 4&deg;C room precipitated phages: centrifuged for 20
+
<div style="margin-left: 40px;"><small>-Over night
 +
in 4&deg;C room precipitated phages: centrifuged for 20
min at 5000rpm and 4&deg;C<br />
min at 5000rpm and 4&deg;C<br />
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet
Line 1,489: Line 1,690:
13000rpm<br />
13000rpm<br />
-Decant the supernatant into new eppis, precipitate with
-Decant the supernatant into new eppis, precipitate with
-
170&micro;l PEG/NaCl and leave for 1 hour on ice<br />
+
170&micro;l PEG/NaCl and leave for 1 hour on ice</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Miniprep
   <li>Miniprep
- pJS 419-his-dig-split-foka (clon 1+2)</li>
- pJS 419-his-dig-split-foka (clon 1+2)</li>
</ul>
</ul>
-
- Glycerolstock of clon1 and 2<br />
+
<div style="margin-left: 40px;"><small>-
 +
Glycerolstock of clon1 and 2<br />
- 300&micro;l Glycerol<br />
- 300&micro;l Glycerol<br />
- 700 &micro;l culture<br />
- 700 &micro;l culture<br />
Line 1,502: Line 1,705:
pJS 419-his-dig-split-foka-clon1= 509.5 ng/&micro;l<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 />
pJS 419-his-dig-split-foka-clon2 =468 ng/&micro;l<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml
   <li>Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml
Tet, over
Tet, over
Line 1,510: Line 1,714:
   <li>send to sequencing:</li>
   <li>send to sequencing:</li>
</ul>
</ul>
-
pMA Dig Plasmidprep 22.07.09 Timo1<br />
+
<div style="margin-left: 40px;"><small>pMA Dig
 +
Plasmidprep 22.07.09 Timo1<br />
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br />
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br />
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br />
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br />
Line 1,519: Line 1,724:
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8<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 clone2 Timo9<br />
-
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12<br />
+
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;concentrated pET39b+ ssDNA via sodiumacetat and
   <li>&nbsp;concentrated pET39b+ ssDNA via sodiumacetat and
ethanol precipitation
ethanol precipitation
Line 1,533: Line 1,739:
   </li>
   </li>
</ul>
</ul>
-
<h3>10.10.09 Julia, Caro, Laura, Christoph</h3>
+
<h3 style="margin-left: 40px;">10.10.09 Julia, Caro,
-
<br />
+
Laura, Christoph</h3>
-
<ul>
+
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>Measured OD600 of over night ER2738 culture abs.1)0.218
   <li>Measured OD600 of over night ER2738 culture abs.1)0.218
(1:10);2)0.23
(1:10);2)0.23
Line 1,559: Line 1,767:
- pBADQuick clones 1-6</li>
- pBADQuick clones 1-6</li>
</ul>
</ul>
 +
<div style="margin-left: 40px;"><small>
- pBADT4 clones 1-6<br />
- pBADT4 clones 1-6<br />
- pMAYFP clone 1-2<br />
- pMAYFP clone 1-2<br />
Line 1,564: Line 1,773:
- pMASplitlinker clones 1-2<br />
- pMASplitlinker clones 1-2<br />
- HisTag clones 1-2 (but test digest was negative -&gt; thrown away)<br />
- HisTag clones 1-2 (but test digest was negative -&gt; thrown away)<br />
-
&lt;
+
</small><br />
-
<br />
+
</div>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>digest for recloning of pMA constructs:
   <li>digest for recloning of pMA constructs:
pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka
pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka
Line 1,573: Line 1,782:
pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10&micro;l</li>
pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10&micro;l</li>
</ul>
</ul>
-
Enzymes: 1&micro;l of NgoMIVI and 1.5&micro;l PstI<br />
+
<div style="margin-left: 40px;"><small>Enzymes:
 +
1&micro;l of NgoMIVI and 1.5&micro;l PstI<br />
Buffer: 3&micro;l buffer 1<br />
Buffer: 3&micro;l buffer 1<br />
BSA (10fold): 3&micro;l<br />
BSA (10fold): 3&micro;l<br />
Line 1,583: Line 1,793:
Buffer: 3&micro;l buffer 1<br />
Buffer: 3&micro;l buffer 1<br />
BSA (10fold): 3&micro;l<br />
BSA (10fold): 3&micro;l<br />
-
Water: 10,5&micro;l<br />
+
Water: 10,5&micro;l</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>PCR assembly with pMADsbA, different approaches (Taq
   <li>PCR assembly with pMADsbA, different approaches (Taq
Polymerase or
Polymerase or
Line 1,598: Line 1,809:
   <li>ligation of -pMAStrepDigShortLiFoka</li>
   <li>ligation of -pMAStrepDigShortLiFoka</li>
</ul>
</ul>
-
-pMAStrepDigMiddleLiFoka<br />
+
<div style="margin-left: 40px;"><small>-pMAStrepDigMiddleLiFoka<br />
-pMAStrepDigLongLiFoka<br />
-pMAStrepDigLongLiFoka<br />
-pExStrepDigShortLiFoka<br />
-pExStrepDigShortLiFoka<br />
Line 1,617: Line 1,828:
vector: 3&micro;l <br />
vector: 3&micro;l <br />
Ouick ligase buffer: 10&micro;l <br />
Ouick ligase buffer: 10&micro;l <br />
-
Quick ligase: 1&micro;l <br />
+
Quick ligase: 1&micro;l </small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>transformation of ligations in XBL on LB/Amp/1%glucose
   <li>transformation of ligations in XBL on LB/Amp/1%glucose
plates
plates
Line 1,629: Line 1,841:
picture</li>
picture</li>
</ul>
</ul>
-
<table style="text-align: left; width: 389px; height: 342px;"
+
<table
 +
style="text-align: left; width: 389px; height: 342px; margin-left: 40px;"
  border="0" cellpadding="0" cellspacing="0">
  border="0" cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
Line 1,638: Line 1,851:
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td>gele of the AGO pETb+ ssDNA cleavage assay. Lanes:
+
       <td><small>gele of the AGO pETb+ ssDNA cleavage
-
Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA</td>
+
assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA</small></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
 +
<div style="margin-left: 40px;"><br />
<br />
<br />
-
<br />
+
</div>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>cotransformation of pExHisFluASplitFoki and
   <li>cotransformation of pExHisFluASplitFoki and
pJSStrepDigSplitFoka in
pJSStrepDigSplitFoka in
Line 1,655: Line 1,869:
   </li>
   </li>
</ul>
</ul>
-
<h3> 11.10.09, Timo, Hannes, Max, Anika</h3>
+
<h3 style="margin-left: 40px;"> 11.10.09, Timo, Hannes,
-
<br />
+
Max, Anika</h3>
-
<ul>
+
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Digestion of&nbsp;</li>
   <li>&nbsp;Digestion of&nbsp;</li>
</ul>
</ul>
-
1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI<br />
+
<div style="margin-left: 40px;"><small>1.pEXHisFluA
 +
(27.09.09, Klon1), XbaI - PstI<br />
2.pMAFos_HlsbZip, XbaI - AgI<br />
2.pMAFos_HlsbZip, XbaI - AgI<br />
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br />
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br />
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br />
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br />
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br />
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br />
-
6.pMA_BB057, XbaI - PstI (01.10.09)<br />
+
6.pMA_BB057, XbaI - PstI (01.10.09)</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>gel extraction of
   <li>gel extraction of
- pMA</li>
- pMA</li>
</ul>
</ul>
-
- pEx<br />
+
<div style="margin-left: 40px;"><small>- pEx<br />
- ...
- ...
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Testdigestion of&nbsp;</li>
   <li>&nbsp;Testdigestion of&nbsp;</li>
</ul>
</ul>
-
7.pMA_YFP 2 (10.10.09, Caro)<br />
+
<div style="margin-left: 40px;"><small>7.pMA_YFP 2
 +
(10.10.09, Caro)<br />
8.pMA_CFP (10.10.09, Caro)<br />
8.pMA_CFP (10.10.09, Caro)<br />
9.pMASplitLi1 (10.10.09, Caro)<br />
9.pMASplitLi1 (10.10.09, Caro)<br />
10.pMASplitLi2 (10.10.09, Caro)<br />
10.pMASplitLi2 (10.10.09, Caro)<br />
-
...image.. <br />
+
...image.. </small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;1% Agarosegel of constructs above
   <li>&nbsp;1% Agarosegel of constructs above
   </li>
   </li>
Line 1,701: Line 1,922:
   </li>
   </li>
</ul>
</ul>
-
<h3> 12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia,
+
<h3 style="margin-left: 40px;"> 12.10.09, Laura, Caro,
 +
Christoph, Anika, Hannes, Julia,
Timo, Gerrit</h3>
Timo, Gerrit</h3>
-
<br />
+
<div style="margin-left: 40px;"><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;protein expression of pEx_DsbA_StrepDigSplitFoka
   <li>&nbsp;protein expression of pEx_DsbA_StrepDigSplitFoka
(periplasm) in
(periplasm) in
Line 1,715: Line 1,938:
Enzymes: 1&micro;l of NgoMIVI and 1.5&micro;l PstI</li>
Enzymes: 1&micro;l of NgoMIVI and 1.5&micro;l PstI</li>
</ul>
</ul>
-
Buffer: 3&micro;l buffer 1<br />
+
<div style="margin-left: 40px;"><small>Buffer:
 +
3&micro;l buffer 1<br />
BSA (100fold): 0.5&micro;l<br />
BSA (100fold): 0.5&micro;l<br />
Water: 14.5&micro;l<br />
Water: 14.5&micro;l<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>&nbsp;Plasmidprep. of: </li>
   <li>&nbsp;Plasmidprep. of: </li>
</ul>
</ul>
-
1.pMAFokA (old)<br />
+
<div style="margin-left: 40px;"><small>1.pMAFokA
 +
(old)<br />
2.pMA-CAT Ndelta4 (MQII)1<br />
2.pMA-CAT Ndelta4 (MQII)1<br />
3.pMA-CAT Ndelta4 (MQII)2<br />
3.pMA-CAT Ndelta4 (MQII)2<br />
Line 1,729: Line 1,955:
6.pMA-CAT Ndelta4 (MQII)5<br />
6.pMA-CAT Ndelta4 (MQII)5<br />
7.pMA-CAT Ndelta4 (MQII)6<br />
7.pMA-CAT Ndelta4 (MQII)6<br />
-
8.pMAFoka clone 1 from 04.10.<br />
+
8.pMAFoka clone 1 from 04.10.</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Made new BB-AGO PCR using digested AGO Gene without the
   <li>Made new BB-AGO PCR using digested AGO Gene without the
vector-&gt; still no expected bands to be seen...
vector-&gt; still no expected bands to be seen...
Line 1,745: Line 1,972:
   <li>Send to sequencing:[[Protocols#DNA_Sequencing]]</li>
   <li>Send to sequencing:[[Protocols#DNA_Sequencing]]</li>
</ul>
</ul>
-
Julia 1-6:<br />
+
<div style="margin-left: 40px;"><small>Julia 1-6:<br />
1)pBADQuick clone 3<br />
1)pBADQuick clone 3<br />
2)pBAD T4 clone 2<br />
2)pBAD T4 clone 2<br />
Line 1,754: Line 1,981:
<br />
<br />
Gerrit1: pMA_cat-Nd4
Gerrit1: pMA_cat-Nd4
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for
   <li>Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for
Panning
Panning
Line 1,771: Line 1,999:
-&gt;analytical gel (see picture)</li>
-&gt;analytical gel (see picture)</li>
</ul>
</ul>
-
-&gt;didn't work again<br />
+
<div style="margin-left: 40px;"><small>
 +
-&gt;didn't work again</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>poured IPTG/XGal plates for in vivo plaque assay test run
   <li>poured IPTG/XGal plates for in vivo plaque assay test run
   </li>
   </li>
   <li>transformation of:</li>
   <li>transformation of:</li>
</ul>
</ul>
-
- pBAD_FokA (Quick)<br />
+
<div style="margin-left: 40px;"><small>- pBAD_FokA
 +
(Quick)<br />
- pBAD_CAT(Quick)<br />
- pBAD_CAT(Quick)<br />
- pBAD_FokA (T4)<br />
- pBAD_FokA (T4)<br />
Line 1,786: Line 2,017:
into BL21 de3 gold<br />
into BL21 de3 gold<br />
Plates: LB+ AMP +1%Glucose<br />
Plates: LB+ AMP +1%Glucose<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>cultivation of
   <li>cultivation of
pma strep dig split foka</li>
pma strep dig split foka</li>
</ul>
</ul>
-
pma long linker<br />
+
<div style="margin-left: 40px;"><small>pma long
-
pma his flua split foki<br />
+
linker<br />
-
<h3> 13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia</h3>
+
pma his flua split foki</small><br />
-
<br />
+
</div>
-
<ul>
+
<h3 style="margin-left: 40px;"> 13.10.09, Laura, Caro,
 +
Christoph,Manu, Hannes, Julia</h3>
 +
<div style="margin-left: 40px;"><br />
 +
</div>
 +
<ul style="margin-left: 40px;">
   <li>Miniprep
   <li>Miniprep
-pMA-fos 1</li>
-pMA-fos 1</li>
</ul>
</ul>
-
-pMA-fos 2<br />
+
<div style="margin-left: 40px;"><small>-pMA-fos 2<br />
-pMA his flua split fokI<br />
-pMA his flua split fokI<br />
- pMA strep dig split foka <br />
- pMA strep dig split foka <br />
-
-pMA long linker<br />
+
-pMA long linker</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>testdigest of Plasmidprep from today
   <li>testdigest of Plasmidprep from today
15 &micro;l for 6&micro;l loading dye
15 &micro;l for 6&micro;l loading dye
-buffer 2 1&micro;l</li>
-buffer 2 1&micro;l</li>
</ul>
</ul>
-
-xbaI 0.5&micro;l<br />
+
<div style="margin-left: 40px;"><small>-xbaI
 +
0.5&micro;l<br />
-pstI 0.75&micro;l<br />
-pstI 0.75&micro;l<br />
-DNA 2 &micro;l<br />
-DNA 2 &micro;l<br />
Line 1,816: Line 2,054:
--&gt; Gelbild
--&gt; Gelbild
- Glycerolstocks are in in box of 5/10/2009
- Glycerolstocks are in in box of 5/10/2009
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>made electrocompetent XL1 Blue cotransformed cells
   <li>made electrocompetent XL1 Blue cotransformed cells
(pExHisFluASplitFoki and pJS419HisDigSplitFoka)
(pExHisFluASplitFoki and pJS419HisDigSplitFoka)
Line 1,829: Line 2,068:
and M13 ssDNA at 1.7kV</li>
and M13 ssDNA at 1.7kV</li>
</ul>
</ul>
-
- 1.5h on 37&deg;C shaker at 750rpm<br />
+
<div style="margin-left: 40px;"><small>- 1.5h on
 +
37&deg;C shaker at 750rpm<br />
- precipitation of phages<br />
- precipitation of phages<br />
- infection of ER2738 with different dilutions of phages<br />
- infection of ER2738 with different dilutions of phages<br />
- mixed cells with Top agar and plated them on IPTG/XGAL plates
- mixed cells with Top agar and plated them on IPTG/XGAL plates
-
-&gt; 37&deg; shaker<br />
+
-&gt; 37&deg; shaker</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>inoculation of
   <li>inoculation of
-pMASplitFoka clone 1 from prep 04.10.09</li>
-pMASplitFoka clone 1 from prep 04.10.09</li>
</ul>
</ul>
-
-pMAStrepDig clone 2 from prep 25.09.09<br />
+
<div style="margin-left: 40px;"><small>-pMAStrepDig
 +
clone 2 from prep 25.09.09<br />
-pMAMiddleFoka clone 2 from prep 04.10.09<br />
-pMAMiddleFoka clone 2 from prep 04.10.09<br />
-pMAShortFoka clone 2 from prep 04.10.09<br />
-pMAShortFoka clone 2 from prep 04.10.09<br />
Line 1,851: Line 2,093:
-pMADsbAStrepDigSplitFoka 13.10.09<br />
-pMADsbAStrepDigSplitFoka 13.10.09<br />
-pExFosSplitFoka 13.10.09<br />
-pExFosSplitFoka 13.10.09<br />
-
-pMAFosSplitFoka 13.10.09<br />
+
-pMAFosSplitFoka 13.10.09</small><br />
<br />
<br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>protein purification of DsbA_StrepDigSplitFoka with Strep
   <li>protein purification of DsbA_StrepDigSplitFoka with Strep
column
column
Line 1,860: Line 2,103:
- pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI</li>
- pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI</li>
</ul>
</ul>
-
- pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI<br />
+
<div style="margin-left: 40px;"><small>-
 +
pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI<br />
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI =&gt;
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI =&gt;
wasn't ok on the gel, inoculated clone 1<br />
wasn't ok on the gel, inoculated clone 1<br />
Line 1,872: Line 2,116:
- pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI
- pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI
- pMA (01.10., c=500ng/&micro;l) with EcoRI + SpeI
- pMA (01.10., c=500ng/&micro;l) with EcoRI + SpeI
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>ligations:
   <li>ligations:
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with EcoRI +
Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with EcoRI +
SpeI</li>
SpeI</li>
</ul>
</ul>
-
Insert: 6 &micro;l LongLinker-FokA (10.09., clone 1) with EcoRI +
+
<div style="margin-left: 40px;"><small>Insert: 6
 +
&micro;l LongLinker-FokA (10.09., clone 1) with EcoRI +
SpeI<br />
SpeI<br />
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
Buffer: 10 &micro;l, QuickLigase Buffer NEB<br />
Line 1,945: Line 2,191:
Buffer: 14 &micro;l, QuickLigase Buffer NEB<br />
Buffer: 14 &micro;l, QuickLigase Buffer NEB<br />
QuickLigase: 1 &micro;l<br />
QuickLigase: 1 &micro;l<br />
 +
</small><br />
<br />
<br />
-
<br />
+
</div>
-
<ul>
+
<ul style="margin-left: 40px;">
   <li>did transformations of the ligations in RV308, plated on
   <li>did transformations of the ligations in RV308, plated on
LB-Amp + 1%
LB-Amp + 1%
Line 1,954: Line 2,201:
   <li>ELISA -Blocking</li>
   <li>ELISA -Blocking</li>
</ul>
</ul>
-
-Washing with TBST-EDTA 5x<br />
+
<div style="margin-left: 40px;"><small>Washing with
-
-Loaded immunotubes (1.5ml) and wells 100&micro;l each with 0.5mM
+
TBST-EDTA 5x<br />
 +
Loaded immunotubes (1.5ml) and wells 100&micro;l each with 0.5mM
biotin-target DNA<br />
biotin-target DNA<br />
-
-Incubation at 20&deg;C in shaking<br />
+
Incubation at 20&deg;C in shaking<br />
-
-Incubatet Phages with Oligo for 30min at 55&deg;c waterbath<br />
+
Incubatet Phages with Oligo for 30min at 55&deg;c waterbath<br />
-
-Washing of wells and immunotubes with TBST-EDTA 5x<br />
+
Washing of wells and immunotubes with TBST-EDTA 5x<br />
-
-Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1
+
Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1
phage+Oligo and 1+control as a control<br />
phage+Oligo and 1+control as a control<br />
-
-Incubation in shaker at 20&deg;C<br />
+
Incubation in shaker at 20&deg;C<br />
-
-Loaded immunotubes (each 1.5 ml) with 50&micro;l P1,
+
Loaded immunotubes (each 1.5 ml) with 50&micro;l P1,
25&micro;l 449Phage+ Oligo in TBST-EDTA<br />
25&micro;l 449Phage+ Oligo in TBST-EDTA<br />
-
-Incubation in shaker at 20&deg;C<br />
+
Incubation in shaker at 20&deg;C<br />
-
-Washing of wells and immunotubes with TBST-EDTA 5x
+
Washing of wells and immunotubes with TBST-EDTA 5x
-Loaded 100&micro;l/well anti-M13 ab(1:1000), incubated for 1 hour
-Loaded 100&micro;l/well anti-M13 ab(1:1000), incubated for 1 hour
at 20&deg;C in shaker<br />
at 20&deg;C in shaker<br />
-
-Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20
+
Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20
min, decanted supernatant into eppis, washed with TBS 3x<br />
min, decanted supernatant into eppis, washed with TBS 3x<br />
-
-2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted
+
2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted
supernatant into eppis, washed with TBS-T 3x<br />
supernatant into eppis, washed with TBS-T 3x<br />
-
-3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis<br />
+
3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis<br />
-
<br />
+
</small><br />
-
<ul>
+
</div>
 +
<ul style="margin-left: 40px;">
   <li>Made new "AGO BB PCR" this time just using the
   <li>Made new "AGO BB PCR" this time just using the
ad-on-tail-primer
ad-on-tail-primer
Line 1,994: Line 2,243:
(5mM), then TBST-MnCl2 (5mM) and then with DNase I
(5mM), then TBST-MnCl2 (5mM) and then with DNase I
   </li>
   </li>
 +
<li>Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence</li>
</ul>
</ul>
-
<div class="cleared"></div>
+
 
 +
<h3 style="margin-left: 40px;"> 14.10.09, Laura, Caro,
 +
Christoph, Hannes, Julia, Anika</h3>
 +
<ul>
 +
  <li>&nbsp;Made LB-medium, 0,7% LB-Agar, 50% Glucose
 +
* Test expression (15ml) of pBAD_CAT and pBAD_FokA in BL21de3
 +
  </li>
 +
</ul>
 +
<table
 +
style="border-collapse: collapse; table-layout: fixed; width: 659pt;"
 +
border="1" cellpadding="0" cellspacing="0"
 +
width="879">
 +
  <tbody>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="height: 15pt; width: 282pt;" height="20"
 +
width="376"><small>OD measurements</small></td>
 +
      <td class="xl6310961"
 +
style="border-left: medium none; width: 89pt;" width="119"><small>1
 +
hour before</small></td>
 +
      <td class="xl6310961"
 +
style="border-left: medium none; width: 73pt;" width="97"><small>induction</small></td>
 +
      <td class="xl6310961"
 +
style="border-left: medium none; width: 80pt;" width="107"><small>45min</small></td>
 +
      <td class="xl6310961"
 +
style="border-left: medium none; width: 68pt;" width="91"><small>1.5h</small></td>
 +
      <td class="xl6310961"
 +
style="border-left: medium none; width: 67pt;" width="89"><small>2h
 +
45min</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>&nbsp;</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>induction</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>&nbsp;</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>&nbsp;</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>&nbsp;</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>&nbsp;</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>1.
 +
clone1 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.1</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.74</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.19</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.25</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.34</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>2.
 +
clone1 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.1</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.67</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.08</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.94</small></td>
 +
      <td class="xl6410961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.20</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>3.
 +
clone 1 pBAD_CAT control</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.07</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.72</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.08</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.24</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.23</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>4.
 +
clone2 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.07</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.63</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.96</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.11</small></td>
 +
      <td class="xl6410961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.00</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>5.
 +
clone2 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.07</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.66</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.06</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.25</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.21</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>6.
 +
clone2 pBAD_CAT control</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.08</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.72</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.11</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.33</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.25</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>7.
 +
clone1 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.07</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.67</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.13</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.26</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.07</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>8.
 +
clone1 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.09</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.67</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.21</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.28</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.22</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>9.
 +
clone1 pBAD_Foka control</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.09</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.7</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.11</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.38</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.31</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>10.
 +
clone2 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.07</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.6</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.93</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.16</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.87</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>11.
 +
clone2 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.07</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.62</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.94</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.18</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.93</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl6310961"
 +
style="border-top: medium none; height: 15pt;" height="20"><small>12.
 +
clone2 pBAD_Foka control</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.01</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>0.64</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.04</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>1.24</small></td>
 +
      <td class="xl6310961"
 +
style="border-top: medium none; border-left: medium none;"><small>2.27</small></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<br>
 +
<div style="margin-left: 40px;"><small>--&gt; no
 +
differences between induced and control cells
 +
<br>
 +
<br>
 +
</small>
 +
<table style="text-align: left; width: 750px;" border="1"
 +
cellpadding="2" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><small><img
 +
style="width: 400px; height: 400px;" alt=""
 +
src="https://static.igem.org/mediawiki/2009/7/70/Freiburg09_141009_pg_testexpression1015.jpg"></small></td>
 +
      <td><img style="width: 400px; height: 400px;"
 +
alt=""
 +
src="https://static.igem.org/mediawiki/2009/1/19/Freiburg09_141009_pg_testexpression2016.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><small>SDS gel of testexpression with <b>pBAD_CAT</b>
 +
in BL21de3; lanes: NEB
 +
prestained protein marker broad range, clone1 pBAD_CAT+0.6mM IPTG+0.2%
 +
Arabinose, clone1 pBAD_CAT control, clone1 pBAD_CAT+0.6mM IPTG+0.002%
 +
Arabinose, empty lane, clone2 pBAD_CAT+0.6mM IPTG+0.2% Arabinose,
 +
clone2 pBAD_CAT control, clone2 pBAD_CAT+0.6mM IPTG+0.002% Arabinose,
 +
empty lane, NEB prestained protein marker borad range</small></td>
 +
      <td><small>SDS gel of testexpression with <b>pBAD_Foka</b>
 +
in BL21de3; lanes: NEB
 +
prestained protein marker broad range, clone1 pBAD_Foka+0.6mM IPTG+0.2%
 +
Arabinose, clone1 pBAD_Foka control, clone1 pBAD_Foka+0.6mM IPTG+0.002%
 +
Arabinose, empty lane, clone2 pBAD_Foka+0.6mM IPTG+0.2% Arabinose,
 +
clone2 pBAD_Foka control, clone2 pBAD_Foka+0.6mM IPTG+0.002% Arabinose</small></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<small><br>
 +
</small></div>
 +
<div style="margin-left: 40px;"><br>
</div>
</div>
 +
<br>
 +
<ul>
 +
  <li>&nbsp;SDS gel of DsbA_StrepDigSplitFoka purification
 +
from 13.10.09</li>
 +
</ul>
 +
<div style="margin-left: 40px;">
 +
<table style="text-align: left; width: 750px;" border="1"
 +
cellpadding="2" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><img style="width: 400px; height: 400px;"
 +
alt=""
 +
src="https://static.igem.org/mediawiki/2009/d/df/Freiburg09_141009_pg_pEx_DsbA_StrepDigSplitFoka_Elution017.jpg"></td>
 +
      <td><img style="width: 400px; height: 400px;"
 +
alt=""
 +
src="https://static.igem.org/mediawiki/2009/d/de/Freiburg09_141009_pg_pEx_DsbA_StrepDigSplitFoka2018.jpg"></td>
 +
    </tr>
 +
    <tr>
 +
      <td><small>SDS gel of protein expression and
 +
purification with Streptag column of <b>pEx_DsbA_StrepDigSplitFoka</b>;
 +
lanes: NEB prestained protein marker broad range, elution fraction 1,
 +
elution fraction 2, elution fraction 3, elution fraction 4, elution
 +
fraction 5, elution fraction 6, elution fraction 7, elution fraction 8,
 +
elution fraction 9</small></td>
 +
      <td><small>SDS gel of protein expression and
 +
purification with Streptag column of <b>pEx_DsbA_StrepDigSplitFoka</b>;
 +
lanes: NEB prestained protein marker broad range, flow through fraction
 +
1, flow through fraction 2, flow through 3, flow through fraction 4,
 +
washing fraction 1, washing fraction 2, washing fraction 3, unpurified
 +
periplasm extract (frozen at -80&deg;C)</small></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<br>
</div>
</div>
 +
<ul>
 +
  <li>Plasmidpreparation&nbsp;</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>1 =
 +
pEX-fos-split-fokA-K1<br>
 +
2 = pEX-fos-split-fokA-K2<br>
 +
3 = pMA-DsbA-strep-dig-split-fokA-K1<br>
 +
4 = pMA-DsbA-strep-dig-split-fokA-K2<br>
 +
5 = pMA-DsbA-his-dig-split-fokA-K1<br>
 +
6 = pMA-DsbA-his-dig-split-fokA-K2<br>
 +
7 = pMA-fos-split-fokA-K1<br>
 +
8 = pMA-fos-split-fokA-K2<br>
 +
9 = pBAD-fokA-K1<br>
 +
10= pBAD-fokA-K2<br>
 +
11 = pBAD-CAT-K1<br>
 +
12= pBAD-CAT-K2<br>
 +
13= pMA-shortlinker-fokA<br>
 +
14= pMA-longlinkerFoka<br>
 +
15= pMA-strep-dig<br>
 +
16= pMA-middlelinker-fokA<br>
 +
18=pMA-split-fokA<br>
 +
-&gt;sequencing of clones 1 and 2 of pBAD_Foka</small><br>
</div>
</div>
-
<br />
+
<ul>
 +
  <li>Pellets of:&nbsp;</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>19=pMA-fokA <br>
 +
20=pEX-DsbA-strep-dig-split-fokA <br>
 +
21=pEX-strep-fluA(3.10) </small><br>
</div>
</div>
 +
<ul>
 +
  <li>digest1:
 +
- pBAD-iGEM(Midi 11.10)</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>-
 +
pMA-DsbA-his-dig-split-fokA (Plasmidprep 14.10 Nr5)<br>
 +
- pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4)<br>
 +
</small></div>
 +
<small><br>
 +
</small>
 +
<div style="margin-left: 40px;"><small>-10&micro;lDNA<br>
 +
-3&micro;l buffer3<br>
 +
-0.5&micro;lBSA(100fold)<br>
 +
-14.5&micro;l water
 +
-1&micro;l EcoRI<br>
 +
-1&micro;l PstI<br>
 +
- loaded on 1% agagel
 +
</small><br>
</div>
</div>
-
<div class="cleared"></div>
+
<ul>
-
<div class="art-Footer">
+
  <li>digest2:
 +
- pMA-fos-split-fokA (Plasmidprep 14.10 Nr7)</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>-
 +
pEX-fos-split-fokA-K1<br>
 +
<br>
 +
-10&micro;lDNA<br>
 +
-3&micro;l buffer4<br>
 +
-0.5&micro;lBSA(100fold)<br>
 +
-14.5&micro;l water
 +
-1&micro;l xbaI<br>
 +
-1&micro;l NgoNIV</small>
 +
<br>
 +
<br>
 +
</div>
 +
<div style="margin-left: 40px;">
 +
loaded on 1% agagel
 +
<br>
 +
</div>
 +
<ul>
 +
  <li>digest3:
 +
-pMA-DsbA-clone2 (5.10)</li>
 +
</ul>
 +
<br>
 +
<div style="margin-left: 40px;"><small>-10&micro;lDNA<br>
 +
-3&micro;l buffer4<br>
 +
-0.5&micro;lBSA(100fold)<br>
 +
-14&micro;l water
 +
-1&micro;l xbaI<br>
 +
-1.5&micro;l AgeI<br>
 +
loaded on 3% agagel
 +
*Gelextraction
 +
-pBAD iGEM<br>
 +
-DsbA-his-dig-split-foka<br>
 +
-DsbA-strep-dig-split-foka<br>
 +
-pMA-fos-dig-split-foka<br>
 +
-pEX-fos-split-foka<br>
 +
--&gt; Picture</small><br>
 +
</div>
 +
<ul>
 +
  <li>Ligation
 +
-pBAD-DsbA-his-dig-split-fokA</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>-pBAD-DsbA-strep-dig-split-fokA<br>
 +
- pMA-fos-dig-split-fokA-DsbA<br>
 +
-pEX-fos-split-fokA-DsbA<br>
 +
-10&micro;l Qick ligase buffer<br>
 +
-1 &micro;l Quick ligase<br>
 +
-6&micro;l Insert<br>
 +
-3&micro;l Vector</small><br>
 +
</div>
 +
<ul>
 +
  <li>&nbsp;Transformation
 +
of the ligation above in X1Blue competent cells</li>
 +
</ul>
 +
<div style="margin-left: 40px;">-100&micro;l and rest
 +
of each transformation were plated on
 +
Amp/Gluc plates
 +
<br>
 +
</div>
 +
<ul>
 +
  <li>made top agar (0.7% agar)
 +
  </li>
 +
  <li>nothing grew on IPTG/Amp plates for test in vivo assay
 +
-&gt; made control assays with</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>-
 +
electroporating
 +
cotransformed XL1Blue with ssM13DNA and plating them
 +
directly to IPTG/Amp cells (no phage precipitation and infection of
 +
ER2738) to test electrocompetence<br>
 +
- infection of ER2738 with phages from lab phage stock and plating them
 +
to IPTG/Amp cells to test plates<br>
 +
-&gt;put plates in 37&deg; room overnight
 +
</small><br>
 +
</div>
 +
<ul>
 +
  <li>inoclutaion of ER2738 from lab stock and our stock in
 +
LB+Tet
 +
-&gt;put them on shaker at 26&deg;C&nbsp;</li>
 +
  <li>&nbsp;XL1 was el. transformed with the ligated
 +
AGO-Phagmid library
 +
(errore-prone PCR of the AGO-G3P construct into an phagmidvektor with
 +
the Tor-A signalling sequence with and without amberstop from
 +
yesterday) and plated everything (4 ml)on two big tubs.&nbsp;</li>
 +
  <li>&nbsp;new PCR for ssDNA from the digested pET39b+
 +
vector to get new target
 +
for an additional AGO assay&nbsp;</li>
 +
  <li>prepaired ELISA to repeat yesterdays AGO-Phage-ELISA
 +
tomorrow (because
 +
yesterdays results were pretty contradictory -&gt; we probably made
 +
a
 +
mistake somewhere)
 +
  </li>
 +
</ul>
 +
<h3 style="margin-left: 40px;"> 15.10.09, Laura, Caro,
 +
Julia, Manu, Timo, Sarah, Hannes, Max, Anika, Christoph</h3>
 +
<ul>
 +
  <li>ELISA:</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>-Blocking:
 +
350&micro;l/well 1% BSA in TBST-EDTA, incubated at
 +
20&deg;C for 2h gently shaking<br>
 +
-5x washing with 200&micro;l/well TBST-EDTA<br>
 +
-Incubation of phages with oligos for 30min in 55&deg;C waterbath<br>
 +
-Incubation of 75&micro;l/well phages for 1h at 20&deg;C gently
 +
shaking<br>
 +
-5x washing with 200&micro;l/well TBST-EDTA<br>
 +
-Antibody: add 75&micro;l/well of anti- M13 antibody (1:1000)<br>
 +
-5x washing with 200&micro;l/well TBST-EDTA<br>
 +
-Substrate: add 75&micro;l/well 1mg/ml ABTS; measure absorbance
 +
immediately and after 3 and 5 min; 405nm</small><br>
 +
</div>
 +
<ul>
 +
  <li>made chemical competent XL1 Blue
 +
*test digests of plasmid preparations from 14.10.09
 +
*seqencing of
 +
- laura1:pExFosSplitFoka clone 2</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>-
 +
laura2:pMAFosSplitFoka clone 2<br>
 +
- laura3:pMADsbAHisDigSplitFoka clone 2<br>
 +
- laura4: pMADsbAStrepDigSplitFoka clone 1</small><br>
 +
</div>
 +
<ul>
 +
  <li>inoculation of - ER2738 from our stock</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>- XL1 blue
 +
from our stock<br>
 +
-&gt; in 50 ml LB+Tetracyclin each, put in 37&deg;C
 +
*inoculation of
 +
-pBAD_Foka clone 1 and 2 from 14.10.09<br>
 +
-pMAFoka clone 1from 04.10.09<br>
 +
-pDsbA clone 2 from 05.10.09</small><br>
 +
</div>
 +
<ul>
 +
  <li>for in vivo assay
 +
- measured pd.7.phage display library concentration new</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>- mixed
 +
1:10^4, 2*10^6, 2*10^7, 2*10^8 dilutons with ER2738 (OD600 of
 +
0.46) and XL1 blue (OD600 of 0.54) each<br>
 +
- let mixtures stand for 10min (infection time)<br>
 +
- mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal
 +
plates<br>
 +
- put it in 37&deg;C roomin the dark<br>
 +
-pExStrepFluASplitFoki from 08.10.09</small><br>
 +
</div>
 +
<div style="margin-left: 40px;"><small>-&gt;
 +
in 5 ml LB+Ampicillin each, put in 37&deg;C
 +
</small><br>
 +
</div>
 +
<br>
 +
<ul>
 +
  <li>&nbsp;new ssDNA production via pET39b+ digestion
 +
product one-primer-pcr&nbsp;</li>
 +
  <li>&nbsp;elusions from the test panning was given to
 +
xl1-&gt;
 +
infection-&gt;
 +
plated in tet cm -&gt; cells infected with the phagmid should
 +
&nbsp;grow-&gt;tomorrow picking clones and determination of the
 +
phagmidinsert&nbsp;</li>
 +
  <li>new AGO BB pcr usind dmso, the gene cut out of the vektor
 +
and only the
 +
ad-on-tail-primers&nbsp;</li>
 +
  <li>starter cultures of AGO Aa and AGO Tt in BL21de3 in
 +
DYT+Kan&nbsp;</li>
 +
  <li>starter cultures of pEx-His-Fos-Split-Foka in RV308 in
 +
DYT+Amp+1%
 +
Glucose
 +
  </li>
 +
</ul>
 +
<h3 style="margin-left: 40px;"> 16.10.09, Laura, Caro,
 +
Julia, Manu, Timo, Sarah,Isabelle, Hannes, Max, Anika, Christoph</h3>
 +
<ul>
 +
  <li>Made 10 L DYT</li>
 +
  <li>Inoculation of elutions from panning: E1#2
 +
(MnCl2+TBS)n&deg;1-20,
 +
E2#1 (MnCl2+TBST)n&deg;1-20, E3#1 (DNAse)n&deg;1-20</li>
 +
</ul>
 +
<h3 style="margin-left: 40px;"> 17.10.09, Laura, Julia,
 +
Manu, Hannes, Gerrit, Christoph</h3>
 +
<ul>
 +
  <li>Expression of Ago Tt in Bl21de3
 +
OD measurement
 +
  </li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small><br>
 +
</small></div>
 +
<table
 +
style="border-collapse: collapse; table-layout: fixed; width: 492pt; margin-left: 40px;"
 +
border="1" cellpadding="0" cellspacing="0"
 +
width="655">
 +
  <tbody>
 +
    <tr>
 +
      <td class="xl15757" style="height: 15pt; width: 96pt;"
 +
height="20" width="128"></td>
 +
      <td class="xl15757" style="width: 116pt;"
 +
width="154"><small>1 hour
 +
before</small></td>
 +
      <td class="xl15757" style="width: 88pt;"
 +
width="117"></td>
 +
      <td class="xl15757" style="width: 48pt;"
 +
width="64"></td>
 +
      <td class="xl15757" style="width: 48pt;"
 +
width="64"></td>
 +
      <td class="xl15757" style="width: 48pt;"
 +
width="64"></td>
 +
      <td class="xl15757" style="width: 48pt;"
 +
width="64"></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"></td>
 +
      <td class="xl15757"><small>induction</small></td>
 +
      <td class="xl15757"><small>time
 +
of induction</small></td>
 +
      <td class="xl15757"><small>1.5
 +
hours</small></td>
 +
      <td class="xl15757"><small>4
 +
hours</small></td>
 +
      <td class="xl15757"><small>6.5
 +
hours</small></td>
 +
      <td class="xl15757"><small>8
 +
hours</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"><small>flask 1</small></td>
 +
      <td class="xl15757" align="right"><small>0.29</small></td>
 +
      <td class="xl15757" align="right"><small>0.53</small></td>
 +
      <td class="xl15757" align="right"><small>0.86</small></td>
 +
      <td class="xl15757" align="right"><small>1.18</small></td>
 +
      <td class="xl15757" align="right"><small>1.32</small></td>
 +
      <td class="xl15757" align="right"><small>1.51</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"><small>flask 2</small></td>
 +
      <td class="xl15757" align="right"><small>0.31</small></td>
 +
      <td class="xl15757" align="right"><small>0.57</small></td>
 +
      <td class="xl15757" align="right"><small>0.91</small></td>
 +
      <td class="xl15757" align="right"><small>1.21</small></td>
 +
      <td class="xl15757" align="right"><small>1.38</small></td>
 +
      <td class="xl15757" align="right"><small>1.49</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"><small>flask 3</small></td>
 +
      <td class="xl15757" align="right"><small>0.32</small></td>
 +
      <td class="xl15757" align="right"><small>0.51</small></td>
 +
      <td class="xl15757" align="right"><small>0.9</small></td>
 +
      <td class="xl15757" align="right"><small>1.31</small></td>
 +
      <td class="xl15757" align="right"><small>1.32</small></td>
 +
      <td class="xl15757" align="right"><small>1.53</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"><small>flask 4</small></td>
 +
      <td class="xl15757" align="right"><small>0.28</small></td>
 +
      <td class="xl15757" align="right"><small>0.49</small></td>
 +
      <td class="xl15757" align="right"><small>0.88</small></td>
 +
      <td class="xl15757" align="right"><small>1.15</small></td>
 +
      <td class="xl15757" align="right"><small>1.33</small></td>
 +
      <td class="xl15757" align="right"><small>1.57</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"><small>flask 5</small></td>
 +
      <td class="xl15757" align="right"><small>0.27</small></td>
 +
      <td class="xl15757" align="right"><small>0.51</small></td>
 +
      <td class="xl15757" align="right"><small>0.89</small></td>
 +
      <td class="xl15757" align="right"><small>1.26</small></td>
 +
      <td class="xl15757" align="right"><small>1.32</small></td>
 +
      <td class="xl15757" align="right"><small>1.48</small></td>
 +
    </tr>
 +
    <tr style="height: 15pt;" height="20">
 +
      <td class="xl15757" style="height: 15pt;"
 +
height="20"><small>flask 6</small></td>
 +
      <td class="xl15757" align="right"><small>0.32</small></td>
 +
      <td class="xl15757" align="right"><small>0.51</small></td>
 +
      <td class="xl15757" align="right"><small>0.83</small></td>
 +
      <td class="xl15757" align="right"><small>1.24</small></td>
 +
      <td class="xl15757" align="right"><small>1.5</small></td>
 +
      <td class="xl15757" align="right"><small>1.61</small></td>
 +
    </tr>
 +
    <tr style="display: none;" height="0">
 +
      <td style="width: 96pt;" width="128"></td>
 +
      <td style="width: 116pt;" width="154"></td>
 +
      <td style="width: 88pt;" width="117"></td>
 +
      <td style="width: 48pt;" width="64"></td>
 +
      <td style="width: 48pt;" width="64"></td>
 +
      <td style="width: 48pt;" width="64"></td>
 +
      <td style="width: 48pt;" width="64"></td>
 +
    </tr>
 +
    <small>
 +
    </small>
 +
  </tbody>
 +
</table>
 +
<small><br>
 +
</small><br>
 +
<ul>
 +
  <li>Dilution from XL1 from OD600 2,8 to OD600 0.07
 +
*Minipreps from : E1#2 (MnCl2+TBS)n&deg;1-20, E2#1
 +
(MnCl2+TBST)n&deg;1-20, E3#1 (DNAse)n&deg;1-20
 +
wich are phagemids we cultured from elutions of panning from 13.10.09</li>
 +
  <li>IPTG/X-Gal
 +
plates with infected ER2738 was blue, with the infected XL1 blue no
 +
blue plaques could be seen -&gt; so infection by phages was
 +
positive
 +
and lots of blue plaques were built up</li>
 +
</ul>
 +
<div style="margin-left: 40px;">
 +
<table style="text-align: left; width: 412px; height: 240px;"
 +
border="1" cellpadding="2" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><a
 +
href="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"><img
 +
style="border: 0px solid ; width: 400px; height: 216px;" alt=""
 +
src="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"></a><a
 +
href="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"
 +
target="_blank"><wbr><wbr></a></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</div>
 +
<div style="margin-left: 40px;">
 +
<ul>
 +
  <li>&nbsp;made
 +
new in vivo test assay with cotransformated, electrocompetent XL1 blue
 +
- transformed M13 ss DNA from 17.08.09 into cells by electroporation at
 +
1.7kV</li>
 +
</ul>
 +
</div>
 +
<div style="margin-left: 80px;"><small>- 1.5h
 +
incubation time on 37&deg;C shaker <br>
 +
- 80, 10 and 5&micro;l of electroporated cells were mixed with
 +
180&micro;l XL1 blue and ER2738 with OD 600 of about 0.5<br>
 +
- incubation time of 10 min at room temperature<br>
 +
- mixing cells with 3ml topagar, vortexing shortly and plating on
 +
IPTG/X-Gal plates<br>
 +
- put in 37&deg;C room overnight</small><br>
 +
</div>
 +
<ul>
 +
  <li>control of electrocompetence of XL1 blue by electroporation
 +
of pET28a
 +
with Kan resistance into XL1 blue and RV respecitvely
 +
-&gt; comparison in the end or if colonies will show up at all will
 +
show if our cotransformated cells are competent at all
 +
*inoculation of
 +
- pMAFoka clone 1 from 04.10.09</li>
 +
</ul>
 +
<div style="margin-left: 40px;"><small>- pMADsbA
 +
clone 2 from 05.10.09<br>
 +
- pBADFoka clone 1 from 15.10.09<br>
 +
- pExStrepFluALongliFoki clone 1 from 08.10.09</small><br>
 +
</div>
 +
<ul>
 +
  <li>miniprep of all clones picked from the elutions of the test
 +
panning to
 +
determine the efficiency of the selection&nbsp;</li>
 +
  <li>new ssDNA via Pet39b+ one primer pcr&nbsp;</li>
 +
  <li>new infection of XL1 cells with the elution from the test
 +
panning-&gt;
 +
this time plated on lb agar with tet cm and GLUC (to suppress the
 +
expression of the phagmid insert)
 +
  </li>
 +
</ul>
 +
<h3 style="margin-left: 40px;"> 18.10.09 Manu</h3>
 +
<ul>
 +
  <li>Expression of Ago Aa at 24&deg;C
 +
*Expression of GST-FosW at 30&deg;C
 +
  </li>
 +
</ul>
 +
<h3 style="margin-left: 40px;"> 19.10.09, Laura, Caro,
 +
Julia, Manu, Timo, Sarah, Isabel, Hannes, Max, Anika, Christoph</h3>
 +
<ul>
 +
  <li>&nbsp;analysis of the geles of the digestion of the
 +
clones picked from the
 +
elution plates revealed not a single clone containing the ago
 +
insert-&gt; possibly the lack of gluc on the plate led to a
 +
selection
 +
against this insert-&gt; new klones picked from the plate with
 +
gluc&nbsp;</li>
 +
  <li>new ago cleavage assay reveald last saturdays promising
 +
results as
 +
false. it seems like it was just two conformations of one ssDNA
 +
molecule&nbsp;</li>
 +
  <li>starter cultures of pEx-His-Fos-Split-Fok_a&nbsp;</li>
 +
  <li>protein purification of GST-FosW with Ni-NTA column
 +
- French pressed, centrifuged 15min at 20000rpm, sonicated and filtered</li>
 +
</ul>
 +
<div style="margin-left: 40px;">- column was washed with
 +
25mM imidazole and eluted with 250mM imidazole
 +
<br>
 +
</div>
 +
<ul>
 +
  <li>&nbsp;in vivo assay retried with shorter incubation
 +
time
 +
  </li>
 +
</ul>
 +
<h3> 20.10.09 Manu, Laura, Anika, Julia, Timo, Sarah, Isabel,
 +
Max, Gerrit, Christoph, Caro, Hannes</h3>
 +
<ul>
 +
  <li>&nbsp;new round of expression with
 +
pEx-His-Fos-Split-Fok_a in BL21de3at
 +
24&deg;C </li>
 +
</ul>
 +
<table x:str=""
 +
style="border-collapse: collapse; table-layout: fixed; width: 344pt;"
 +
border="1" cellpadding="0" cellspacing="0"
 +
width="459">
 +
  <tbody>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995"
 +
style="height: 12.75pt; width: 96pt;" height="17"
 +
width="128"><small>OD measurement</small></td>
 +
      <td class="xl1530995" style="width: 56pt;"
 +
width="75"><small>time of</small></td>
 +
      <td class="xl1530995" style="width: 48pt;"
 +
width="64"><small>2 hours</small></td>
 +
      <td class="xl1530995" style="width: 48pt;"
 +
width="64"><small>3 hours</small></td>
 +
      <td class="xl1530995" style="width: 48pt;"
 +
width="64"><small>3 hours</small></td>
 +
      <td class="xl1530995" style="width: 48pt;"
 +
width="64"><small>4 hours</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small></small></td>
 +
      <td class="xl1530995"><small>induction</small></td>
 +
      <td class="xl1530995"><small></small></td>
 +
      <td class="xl1530995"><small></small></td>
 +
      <td class="xl1530995"><small>45 min</small></td>
 +
      <td class="xl1530995"><small>15 min</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small>flask 1</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>1.3</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>2.63</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.47</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.29</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.99</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small>flask 2</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>1.19</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>2.87</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.87</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.51</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.62</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small>flask 3</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>1.5</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.22</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.45</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>5.03</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>5.41</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small>flask 4</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>1.03</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>2.9</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.21</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>5.11</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>5.88</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small>flask 5</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>0.81</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>2.81</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.76</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.29</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>4.76</small></td>
 +
    </tr>
 +
    <tr style="height: 12.75pt;" height="17">
 +
      <td class="xl1530995" style="height: 12.75pt;"
 +
height="17"><small>flask 6</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>1.52</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>2.72</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.71</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>3.95</small></td>
 +
      <td class="xl1530995" x:num="" align="right"><small>5.17</small></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
 
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<div class="art-Footer-inner">
<div class="art-Footer-text">
<div class="art-Footer-text">

Latest revision as of 12:19, 21 October 2009

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
FokI wildtype cutting
Hybridize M13 ssDNA with Fok control oligos 2 and 3
Volume Reagent
10 µl M13 ssDNA (c = 127 ng/µl, 17.08.09, sample #5.2)
5 µl MgCl2 (5 mM)
5 µl Tris-HCl pH 8 (100 mM)
2 µl Fok control 2 (10 ng/µl; 1 µM)
2 µl Fok control 3 (10 ng/µl; 1 µM)
30 µl

The hybridization was also done with just Fok control 2 and with no oligos at all.
Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).
10 µl of the hybridized DNA were used for a cutting experiment with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI (wildtype Fok from NEB). The digest was incubated for 30 minutes at 37°C.

Agarose gel of FokI cutting M13 ssDNA
Agarose gel of FokI cutting M13 ssDNA
Lane 1: GeneRuler DNA Ladder Mix (Fermentas, 6 µl);
Lane 2: Untreated M13 ssDNA (250 ng);
Lane 3: Digest of M13 ssDNA with FokI and without oligo;
Lane 4: Digest of M13 ssDNA with FokI and with one oligo;
Lane 5: Digest of M13 ssDNA with FokI and two oligos.

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
preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka
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
  • Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence

14.10.09, Laura, Caro, Christoph, Hannes, Julia, Anika

  •  Made LB-medium, 0,7% LB-Agar, 50% Glucose * Test expression (15ml) of pBAD_CAT and pBAD_FokA in BL21de3
OD measurements 1 hour before induction 45min 1.5h 2h 45min
  induction        
1. clone1 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose 0.1 0.74 1.19 1.25 2.34
2. clone1 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose 0.1 0.67 1.08 0.94 2.20
3. clone 1 pBAD_CAT control 0.07 0.72 1.08 1.24 2.23
4. clone2 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose 0.07 0.63 0.96 1.11 2.00
5. clone2 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose 0.07 0.66 1.06 1.25 2.21
6. clone2 pBAD_CAT control 0.08 0.72 1.11 1.33 2.25
7. clone1 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose 0.07 0.67 1.13 1.26 2.07
8. clone1 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose 0.09 0.67 1.21 1.28 2.22
9. clone1 pBAD_Foka control 0.09 0.7 1.11 1.38 2.31
10. clone2 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose 0.07 0.6 0.93 1.16 1.87
11. clone2 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose 0.07 0.62 0.94 1.18 1.93
12. clone2 pBAD_Foka control 0.01 0.64 1.04 1.24 2.27

--> no differences between induced and control cells

SDS gel of testexpression with pBAD_CAT in BL21de3; lanes: NEB prestained protein marker broad range, clone1 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, clone1 pBAD_CAT control, clone1 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, empty lane, clone2 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, clone2 pBAD_CAT control, clone2 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, empty lane, NEB prestained protein marker borad range SDS gel of testexpression with pBAD_Foka in BL21de3; lanes: NEB prestained protein marker broad range, clone1 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, clone1 pBAD_Foka control, clone1 pBAD_Foka+0.6mM IPTG+0.002% Arabinose, empty lane, clone2 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, clone2 pBAD_Foka control, clone2 pBAD_Foka+0.6mM IPTG+0.002% Arabinose



  •  SDS gel of DsbA_StrepDigSplitFoka purification from 13.10.09
SDS gel of protein expression and purification with Streptag column of pEx_DsbA_StrepDigSplitFoka; lanes: NEB prestained protein marker broad range, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, elution fraction 8, elution fraction 9 SDS gel of protein expression and purification with Streptag column of pEx_DsbA_StrepDigSplitFoka; lanes: NEB prestained protein marker broad range, flow through fraction 1, flow through fraction 2, flow through 3, flow through fraction 4, washing fraction 1, washing fraction 2, washing fraction 3, unpurified periplasm extract (frozen at -80°C)

  • Plasmidpreparation 
1 = pEX-fos-split-fokA-K1
2 = pEX-fos-split-fokA-K2
3 = pMA-DsbA-strep-dig-split-fokA-K1
4 = pMA-DsbA-strep-dig-split-fokA-K2
5 = pMA-DsbA-his-dig-split-fokA-K1
6 = pMA-DsbA-his-dig-split-fokA-K2
7 = pMA-fos-split-fokA-K1
8 = pMA-fos-split-fokA-K2
9 = pBAD-fokA-K1
10= pBAD-fokA-K2
11 = pBAD-CAT-K1
12= pBAD-CAT-K2
13= pMA-shortlinker-fokA
14= pMA-longlinkerFoka
15= pMA-strep-dig
16= pMA-middlelinker-fokA
18=pMA-split-fokA
->sequencing of clones 1 and 2 of pBAD_Foka
  • Pellets of: 
19=pMA-fokA
20=pEX-DsbA-strep-dig-split-fokA
21=pEX-strep-fluA(3.10)
  • digest1: - pBAD-iGEM(Midi 11.10)
- pMA-DsbA-his-dig-split-fokA (Plasmidprep 14.10 Nr5)
- pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4)
-10µlDNA
-3µl buffer3
-0.5µlBSA(100fold)
-14.5µl water -1µl EcoRI
-1µl PstI
- loaded on 1% agagel
  • digest2: - pMA-fos-split-fokA (Plasmidprep 14.10 Nr7)
- pEX-fos-split-fokA-K1

-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14.5µl water -1µl xbaI
-1µl NgoNIV

loaded on 1% agagel
  • digest3: -pMA-DsbA-clone2 (5.10)

-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14µl water -1µl xbaI
-1.5µl AgeI
loaded on 3% agagel *Gelextraction -pBAD iGEM
-DsbA-his-dig-split-foka
-DsbA-strep-dig-split-foka
-pMA-fos-dig-split-foka
-pEX-fos-split-foka
--> Picture
  • Ligation -pBAD-DsbA-his-dig-split-fokA
-pBAD-DsbA-strep-dig-split-fokA
- pMA-fos-dig-split-fokA-DsbA
-pEX-fos-split-fokA-DsbA
-10µl Qick ligase buffer
-1 µl Quick ligase
-6µl Insert
-3µl Vector
  •  Transformation of the ligation above in X1Blue competent cells
-100µl and rest of each transformation were plated on Amp/Gluc plates
  • made top agar (0.7% agar)
  • nothing grew on IPTG/Amp plates for test in vivo assay -> made control assays with
- electroporating cotransformed XL1Blue with ssM13DNA and plating them directly to IPTG/Amp cells (no phage precipitation and infection of ER2738) to test electrocompetence
- infection of ER2738 with phages from lab phage stock and plating them to IPTG/Amp cells to test plates
->put plates in 37° room overnight
  • inoclutaion of ER2738 from lab stock and our stock in LB+Tet ->put them on shaker at 26°C 
  •  XL1 was el. transformed with the ligated AGO-Phagmid library (errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence with and without amberstop from yesterday) and plated everything (4 ml)on two big tubs. 
  •  new PCR for ssDNA from the digested pET39b+ vector to get new target for an additional AGO assay 
  • prepaired ELISA to repeat yesterdays AGO-Phage-ELISA tomorrow (because yesterdays results were pretty contradictory -> we probably made a mistake somewhere)

15.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Hannes, Max, Anika, Christoph

  • ELISA:
-Blocking: 350µl/well 1% BSA in TBST-EDTA, incubated at 20°C for 2h gently shaking
-5x washing with 200µl/well TBST-EDTA
-Incubation of phages with oligos for 30min in 55°C waterbath
-Incubation of 75µl/well phages for 1h at 20°C gently shaking
-5x washing with 200µl/well TBST-EDTA
-Antibody: add 75µl/well of anti- M13 antibody (1:1000)
-5x washing with 200µl/well TBST-EDTA
-Substrate: add 75µl/well 1mg/ml ABTS; measure absorbance immediately and after 3 and 5 min; 405nm
  • made chemical competent XL1 Blue *test digests of plasmid preparations from 14.10.09 *seqencing of - laura1:pExFosSplitFoka clone 2
- laura2:pMAFosSplitFoka clone 2
- laura3:pMADsbAHisDigSplitFoka clone 2
- laura4: pMADsbAStrepDigSplitFoka clone 1
  • inoculation of - ER2738 from our stock
- XL1 blue from our stock
-> in 50 ml LB+Tetracyclin each, put in 37°C *inoculation of -pBAD_Foka clone 1 and 2 from 14.10.09
-pMAFoka clone 1from 04.10.09
-pDsbA clone 2 from 05.10.09
  • for in vivo assay - measured pd.7.phage display library concentration new
- mixed 1:10^4, 2*10^6, 2*10^7, 2*10^8 dilutons with ER2738 (OD600 of 0.46) and XL1 blue (OD600 of 0.54) each
- let mixtures stand for 10min (infection time)
- mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal plates
- put it in 37°C roomin the dark
-pExStrepFluASplitFoki from 08.10.09
-> in 5 ml LB+Ampicillin each, put in 37°C

  •  new ssDNA production via pET39b+ digestion product one-primer-pcr 
  •  elusions from the test panning was given to xl1-> infection-> plated in tet cm -> cells infected with the phagmid should  grow->tomorrow picking clones and determination of the phagmidinsert 
  • new AGO BB pcr usind dmso, the gene cut out of the vektor and only the ad-on-tail-primers 
  • starter cultures of AGO Aa and AGO Tt in BL21de3 in DYT+Kan 
  • starter cultures of pEx-His-Fos-Split-Foka in RV308 in DYT+Amp+1% Glucose

16.10.09, Laura, Caro, Julia, Manu, Timo, Sarah,Isabelle, Hannes, Max, Anika, Christoph

  • Made 10 L DYT
  • Inoculation of elutions from panning: E1#2 (MnCl2+TBS)n°1-20, E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20

17.10.09, Laura, Julia, Manu, Hannes, Gerrit, Christoph

  • Expression of Ago Tt in Bl21de3 OD measurement

1 hour before
induction time of induction 1.5 hours 4 hours 6.5 hours 8 hours
flask 1 0.29 0.53 0.86 1.18 1.32 1.51
flask 2 0.31 0.57 0.91 1.21 1.38 1.49
flask 3 0.32 0.51 0.9 1.31 1.32 1.53
flask 4 0.28 0.49 0.88 1.15 1.33 1.57
flask 5 0.27 0.51 0.89 1.26 1.32 1.48
flask 6 0.32 0.51 0.83 1.24 1.5 1.61


  • Dilution from XL1 from OD600 2,8 to OD600 0.07 *Minipreps from : E1#2 (MnCl2+TBS)n°1-20, E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20 wich are phagemids we cultured from elutions of panning from 13.10.09
  • IPTG/X-Gal plates with infected ER2738 was blue, with the infected XL1 blue no blue plaques could be seen -> so infection by phages was positive and lots of blue plaques were built up
  •  made new in vivo test assay with cotransformated, electrocompetent XL1 blue - transformed M13 ss DNA from 17.08.09 into cells by electroporation at 1.7kV
- 1.5h incubation time on 37°C shaker
- 80, 10 and 5µl of electroporated cells were mixed with 180µl XL1 blue and ER2738 with OD 600 of about 0.5
- incubation time of 10 min at room temperature
- mixing cells with 3ml topagar, vortexing shortly and plating on IPTG/X-Gal plates
- put in 37°C room overnight
  • control of electrocompetence of XL1 blue by electroporation of pET28a with Kan resistance into XL1 blue and RV respecitvely -> comparison in the end or if colonies will show up at all will show if our cotransformated cells are competent at all *inoculation of - pMAFoka clone 1 from 04.10.09
- pMADsbA clone 2 from 05.10.09
- pBADFoka clone 1 from 15.10.09
- pExStrepFluALongliFoki clone 1 from 08.10.09
  • miniprep of all clones picked from the elutions of the test panning to determine the efficiency of the selection 
  • new ssDNA via Pet39b+ one primer pcr 
  • new infection of XL1 cells with the elution from the test panning-> this time plated on lb agar with tet cm and GLUC (to suppress the expression of the phagmid insert)

18.10.09 Manu

  • Expression of Ago Aa at 24°C *Expression of GST-FosW at 30°C

19.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Isabel, Hannes, Max, Anika, Christoph

  •  analysis of the geles of the digestion of the clones picked from the elution plates revealed not a single clone containing the ago insert-> possibly the lack of gluc on the plate led to a selection against this insert-> new klones picked from the plate with gluc 
  • new ago cleavage assay reveald last saturdays promising results as false. it seems like it was just two conformations of one ssDNA molecule 
  • starter cultures of pEx-His-Fos-Split-Fok_a 
  • protein purification of GST-FosW with Ni-NTA column - French pressed, centrifuged 15min at 20000rpm, sonicated and filtered
- column was washed with 25mM imidazole and eluted with 250mM imidazole
  •  in vivo assay retried with shorter incubation time

20.10.09 Manu, Laura, Anika, Julia, Timo, Sarah, Isabel, Max, Gerrit, Christoph, Caro, Hannes

  •  new round of expression with pEx-His-Fos-Split-Fok_a in BL21de3at 24°C
OD measurement time of 2 hours 3 hours 3 hours 4 hours
induction 45 min 15 min
flask 1 1.3 2.63 3.47 4.29 3.99
flask 2 1.19 2.87 3.87 4.51 4.62
flask 3 1.5 3.22 4.45 5.03 5.41
flask 4 1.03 2.9 4.21 5.11 5.88
flask 5 0.81 2.81 3.76 4.29 4.76
flask 6 1.52 2.72 3.71 3.95 5.17

contact:  freigem09@googlemail.com

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