Team:Marburg/Notebook:April

From 2013.igem.org

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Notebook: April
+
Notebook: April <html><a href="https://2013.igem.org/Team:Marburg/Notebook:Mai"><img src="https://static.igem.org/mediawiki/2013/7/71/Mr-igem-next-arrow.png" style="float:right;margin-left:5px !important;" alt="Next"></a>&nbsp;<a href="https://2013.igem.org/Team:Marburg/Notebook:March"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html>
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{{:Team:Marburg/Template:ContentStartNav}}<html>
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-
<html>
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<li>1 µl T4 DNA ligase</li>
<li>1 µl T4 DNA ligase</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at RT.</p>
+
<p>The samples are incubated for 2 h at RT.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 46: Line 44:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation into <i>E. coli</i> DH5&alpha; ( 30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, oN 37°C.</p>
+
<p>Transformation into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 88: Line 86:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>98</td>
<td>98</td>
Line 131: Line 129:
<tr>
<tr>
<td>26 µl</td>
<td>26 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 212: Line 210:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Colonies 1-3 + 5 inoculated in 5 ml LB<sub>amp</sub>, oN 37°C.</p>
+
<p>Colonies 1-3 + 5 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 236: Line 234:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.</p>
+
<p>Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 271: Line 269:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 278: Line 276:
<tr>
<tr>
<td>1,5 µl</td>
<td>1,5 µl</td>
-
<td>Primer 13 (10pmol/µl)</td>
+
<td>Primer 13 (10 pmol/µl)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 285: Line 283:
<tr>
<tr>
<td>1,5 µl</td>
<td>1,5 µl</td>
-
<td>Primer 14 (10pmol/µl) </td>
+
<td>Primer 14 (10 pmol/µl) </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>58</td>
<td>58</td>
Line 314: Line 312:
<tr>
<tr>
<td>ad 50 µl</td>
<td>ad 50 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 352: Line 350:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 359: Line 357:
<tr>
<tr>
<td>1,5 µl</td>
<td>1,5 µl</td>
-
<td>Primer 15 (10pmol/µl)</td>
+
<td>Primer 15 (10 pmol/µl)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 366: Line 364:
<tr>
<tr>
<td>1,5 µl</td>
<td>1,5 µl</td>
-
<td>Primer 16 (10pmol/µl) </td>
+
<td>Primer 16 (10 pmol/µl) </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>63</td>
<td>63</td>
Line 395: Line 393:
<tr>
<tr>
<td>ad 50 µl</td>
<td>ad 50 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 422: Line 420:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of products of the second PCR into <i>E. coli</i> DH5&alpha; ( 30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, oN 37°C.</p>
+
<p>Transformation of products of the second PCR into <i>E. coli</i> DH5&alpha; ( 30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 446: Line 444:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>PCR of iBB1 (pre- and suffix), iBB6 (pre- and suffix), and iBB2 (mutagenesis of PvuII-site), second PCR of iBB2 (mutagenesis of EcoRI-site), iBB7 (pre- and suffix) and iBB8 (pre- and suffix). All done with analog mix and program. Primers vary.</p>
+
<p>PCR of iBB1 (pre- and suffix), iBB6 (pre- and suffix), and iBB2 (mutagenesis of <i>Pvu</i>II-site), second PCR of iBB2 (mutagenesis of <i>EcoR</i>I-site), iBB7 (pre- and suffix) and iBB8 (pre- and suffix). All done with analog mix and program. Primers vary.</p>
<table class="pcr">
<table class="pcr">
<colgroup>
<colgroup>
Line 465: Line 463:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 508: Line 506:
<tr>
<tr>
<td>10 µl</td>
<td>10 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 621: Line 619:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of the PCR-product of iBB2 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, oN 37°C.</p>
+
<p>Transformation of the PCR-product of iBB2 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 672: Line 670:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 715: Line 713:
<tr>
<tr>
<td>35 µl</td>
<td>35 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 852: Line 850:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of pSB1C3 J04450 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB<sub>CM</sub>, oN 37°C.</p>
+
<p>Transformation of pSB1C3 J04450 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB<sub>CM</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 893: Line 891:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Some colonies of pJet1.2 iBB2 inoculated in 5 ml LB<sub>amp</sub> and pSB1C3 J04450 inoculated in 5 ml LB<sub>CM</sub>, oN 37°C.</p>
+
<p>Some colonies of pJet1.2 iBB2 inoculated in 5 ml LB<sub>amp</sub> and pSB1C3 J04450 inoculated in 5 ml LB<sub>CM</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 927: Line 925:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 934: Line 932:
<tr>
<tr>
<td>1,5 µl</td>
<td>1,5 µl</td>
-
<td>Primer 15 (10pmol/µl)</td>
+
<td>Primer 15 (10 pmol/µl)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 941: Line 939:
<tr>
<tr>
<td>1,5 µl</td>
<td>1,5 µl</td>
-
<td>Primer 16 (10pmol/µl) </td>
+
<td>Primer 16 (10 pmol/µl) </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>58</td>
<td>58</td>
Line 970: Line 968:
<tr>
<tr>
<td>35 µl</td>
<td>35 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 996: Line 994:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Miniprep of pJet1.2 iBB2 and pSB1C3 J04450 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 25 µl H2O.</p>
+
<p>Miniprep of pJet1.2 iBB2 and pSB1C3 J04450 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,017: Line 1,015:
<li>1 µl <i>Pst</i>I</li>
<li>1 µl <i>Pst</i>I</li>
<li>2 µl buffer O (Fermentas)</li>
<li>2 µl buffer O (Fermentas)</li>
-
<li>13 µl H2O</li>
+
<li>13 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 1,110: Line 1,108:
<li>4 µl <i>Pst</i>I</li>
<li>4 µl <i>Pst</i>I</li>
<li>8 µl buffer O</li>
<li>8 µl buffer O</li>
-
<li>6 µl H2O</li>
+
<li>6 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
</div>
</div>
Line 1,144: Line 1,142:
<li>3,5 µl buffer O</li>
<li>3,5 µl buffer O</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
</div>
</div>
Line 1,179: Line 1,177:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 1,222: Line 1,220:
<tr>
<tr>
<td>35 µl</td>
<td>35 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 1,266: Line 1,264:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of pSB1C3 iBB5 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB<sub>CM</sub>, oN 37°C.</p>
+
<p>Transformation of pSB1C3 iBB5 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB<sub>CM</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,298: Line 1,296:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Inoculation of pSB1C3 J04450 in LB<sub>CM</sub>, oN 37°C.</p>
+
<p>Inoculation of pSB1C3 J04450 in LB<sub>CM</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,322: Line 1,320:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 25 µl H2O.</p>
+
<p>Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,343: Line 1,341:
<li>1 µl <i>Pst</i>I</li>
<li>1 µl <i>Pst</i>I</li>
<li>4 µl buffer O</li>
<li>4 µl buffer O</li>
-
<li>3 µl H2O</li>
+
<li>3 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 1,488: Line 1,486:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 1,531: Line 1,529:
<tr>
<tr>
<td>10 µl</td>
<td>10 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 1,625: Line 1,623:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Miniprep of iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 25 µl H2O.</p>
+
<p>Miniprep of iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,647: Line 1,645:
<li>1 µl <i>Pst</i>I</li>
<li>1 µl <i>Pst</i>I</li>
<li>2 µl buffer O (Fermentas)</li>
<li>2 µl buffer O (Fermentas)</li>
-
<li>13 µl H2O</li>
+
<li>13 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
-
<col width="50%" />
+
<col width="42%" />
-
<col width="50%" />
+
<col width="58%" />
</colgroup>
</colgroup>
<thead>
<thead>
Line 1,663: Line 1,661:
<tr>
<tr>
<td>
<td>
-
<img src="https://static.igem.org/mediawiki/2013/1/11/Gel_2013-04-22_3.png" width="100" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/1/11/Gel_2013-04-22_3.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 1,680: Line 1,678:
<col width="10%" />
<col width="10%" />
<col width="2%" />
<col width="2%" />
-
<col width="50%" />
+
<col width="55%" />
<col width="5%" />
<col width="5%" />
-
<col width="33%" />
+
<col width="28%" />
</colgroup>
</colgroup>
<thead>
<thead>
Line 1,767: Line 1,765:
<li>1 µl <i>Pst</i>I</li>
<li>1 µl <i>Pst</i>I</li>
<li>4 µl buffer O</li>
<li>4 µl buffer O</li>
-
<li>3 µl H2O</li>
+
<li>3 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 2 h at 37° C.</p>
+
<p>The samples were incubated for 2 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 1,783: Line 1,781:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/1/19/Gel_2013-04-22_1b.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 1,795: Line 1,793:
</ul>
</ul>
</p>
</p>
 +
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-3</td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3-iBB2</td>
 +
<th>&nbsp;</th>
 +
<td>660 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4-7</td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3-iBB8</td>
 +
<th>&nbsp;</th>
 +
<td>1404 bp</td>
 +
</tr>
 +
 +
</table>
 +
</p>
 +
<p>We receive  expected fragments for sample 2 and 3.</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2" class="gel-fazit">
 +
<p>2 out of 6 are positive.</p>
 +
</td>
 +
</tr>
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.</p>
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.</p>
</td>
</td>
Line 1,815: Line 1,854:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Ligation of iBB2 and iBB8 into pSB1C3, oN 16° C.</p>
+
<p>Ligation of iBB2 and iBB8 into pSB1C3, overnight at 16 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,839: Line 1,878:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of pSB1C3 iBB2 and pSB1C3 iBB8 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB<sub>CM</sub>, oN 37°C.</p>
+
<p>Transformation of pSB1C3 iBB2 and pSB1C3 iBB8 into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LB<sub>CM</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,879: Line 1,918:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>2 colonies of pSB1C3 iBB2 and iBB8 inoculated in 5 ml LB<sub>CM</sub>, oN 37° C.</p>
+
<p>2 colonies of pSB1C3 iBB2 and iBB8 inoculated in 5 ml LB<sub>CM</sub>, overnight at 37 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,924: Line 1,963:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Original pPhaNR was sent to sequencing using primers imR25/26 for iBB3 and iBB6.</p>
+
<p>Original pPhaNR was sent to sequencing using primers iMR25/26 for iBB3 and iBB6.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,940: Line 1,979:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>PSB1C3 iBB8 K1 and pSB1C3 iBB2 K1-4 were isolated via Miniprep (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 40 l Elutionbuffer.
+
<p>PSB1C3 iBB8 K1 and pSB1C3 iBB2 K1-4 were isolated via Miniprep (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 40 l Elutionbuffer.
</p>
</p>
</div>
</div>
Line 1,962: Line 2,001:
<li>0,5 µl <i>Pst</i>I</li>
<li>0,5 µl <i>Pst</i>I</li>
<li>1 µl Cut Smart buffer</li>
<li>1 µl Cut Smart buffer</li>
-
<li>7 µl H2O</li>
+
<li>7 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 1,978: Line 2,017:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/9/92/Gel_2013-04-26.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 1,986: Line 2,025:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe per 50 ml gel</li>
<li>10 µl RedSafe per 50 ml gel</li>
-
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
+
<li>6 µl GeneRuler&trade; 2-log DNA Ladder (Thermo Scientific)</li>
<li>6x loading buffer (for samples)</li>
<li>6x loading buffer (for samples)</li>
</ul>
</ul>
</p>
</p>
-
<p>IBB8 negative, iBB2 all positive.</p>
+
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3-iBB8</td>
 +
<th>&nbsp;</th>
 +
<td>1404 bp</td>
 +
</tr>
 +
<tr>
 +
<td>3-6</td>
 +
<th>&nbsp;</th>
 +
<td>pSB1C3-iBB2</td>
 +
<th>&nbsp;</th>
 +
<td>660 bp</td>
 +
</tr>
 +
 +
</table>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2" class="gel-fazit">
 +
 +
</td>
 +
</tr>
 +
</td>
</td>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>
 +
<p>IBB8 negative, iBB2 all positive.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 2,100: Line 2,180:
<tr>
<tr>
<td>33 µl</td>
<td>33 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 2,119: Line 2,199:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/8/89/Gel_2013-04-29.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 2,127: Line 2,207:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
-
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
+
<li>6 µl GeneRuler&trade; 2-log DNA Ladder (NEB)</li>
<li>6x loading buffer (for samples)</li>
<li>6x loading buffer (for samples)</li>
</ul>
</ul>
</p>
</p>
-
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.</p>
+
<p>
 +
<table class="gel">
 +
<colgroup>
 +
<col width="10%" />
 +
<col width="2%" />
 +
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2-3</td>
 +
<th>&nbsp;</th>
 +
<td>iBB8, PCR I</td>
 +
<th>&nbsp;</th>
 +
<td>1404 bp</td>
 +
</tr>
 +
<tr>
 +
<td>5-6</td>
 +
<th>&nbsp;</th>
 +
<td>iBB8, PCR II</td>
 +
<th>&nbsp;</th>
 +
<td>1404 bp</td>
 +
</tr>
 +
 +
</table>
 +
</p>
 +
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl bidest. H2O.</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2" class="gel-fazit">
 +
 +
</td>
 +
</tr>
 +
</td>
</td>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>
 +
<p>All positive.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 2,157: Line 2,279:
<li>3,6 µl Cut Smart buffer</li>
<li>3,6 µl Cut Smart buffer</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 2,168: Line 2,290:
</tr>
</tr>
</thead>
</thead>
-
<tbody>
+
</tbody>
-
<tr>
+
</table>
-
<td>
+
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
-
</td>
+
-
<td>
+
-
<p>
+
-
<span class="gel-elc">Gel substances</span>
+
-
<ul class="gel-sub">
+
-
<li>1% Agarose gel</li>
+
-
<li>10 µl RedSafe per 50 ml gel</li>
+
-
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
+
-
<li>6x loading buffer (for samples)</li>
+
-
</ul>
+
-
</p>
+
-
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.</p>
+
-
</td>
+
-
</tr>
+
-
</tbody>
+
-
</table>
+
</div>
</div>
</fieldset>
</fieldset>
Line 2,208: Line 2,313:
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>1 µl T4 DNA ligase</li>
<li>1 µl T4 DNA ligase</li>
-
<li>ad 20 µl H2O</li>
+
<li>ad 20 µl bidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated overnight at 16° C.</p>
+
<p>The samples were incubated overnight at 16 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 2,236: Line 2,341:
<p>The complete ligation sample was mixed with one aliquot of chemo-competent
<p>The complete ligation sample was mixed with one aliquot of chemo-competent
<i>E. coli</i> DH5&alpha; cells (50 µl) and incubated for 30 min on ice.  
<i>E. coli</i> DH5&alpha; cells (50 µl) and incubated for 30 min on ice.  
-
The heatshock was performed sec at 42° C for 60 and cells were then incubated at  
+
The heatshock was performed at 42 °C for 60 sec and cells were then incubated at  
-
37° C for 1 h with 900 µl LB. The sample was concentrated and plated on  
+
37 °C for 1 h with 900 µl LB. The sample was concentrated and plated on  
LB containing chloramphenicol.
LB containing chloramphenicol.
-
<br />The plate was incubated for two days at 28° C .</p>
+
<br />The plate was incubated for two days at 28 °C .</p>
</div>
</div>
</fieldset>
</fieldset>

Latest revision as of 11:13, 27 October 2013

Notebook: April Next Previous

02.04.2013

Ligation
Investigator: Christian
Aim: Construction of BioBricks → Ligation of iBB2 into pJet1.2.
  • 1 µl pJet1.2 (provided already cut)
  • 25 µl PCR-product CAT-cassette
  • 3 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase

The samples are incubated for 2 h at RT.

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation of pJet1.2 iBB2.

Transformation into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

03.04.2013

PCR
Investigator: Christian
Aim: Construction of BioBricks → Control of pJet1.2 iBB2 via colony-PCR.
Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   98 3 min
1 µl Primer fw   98 30 sec
1 µl Primer rv   65 30 sec x26
10 µl Template   72 1 min
1 µl dNTPs   72 7 min
10 µl 5x buffer   4 Hold
26 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-8   Colony PCR iBB2   717 bp

We receive all expected fragments.

Colony PCR is successful.
Inoculation
Investigator: Christian
Aim: Construction of BioBricks → Inoculation of positive pJet1.2 iBB2.

Colonies 1-3 + 5 inoculated in 5 ml LBamp, overnight at 37°C.

04.04.2013

Miniprep
Investigator: Christian
Aim: Construction of BioBricks → Miniprep of pJet1.2 iBB2.

Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Deletion of restriction sites in iBB2 via mutagenesis PCR.

Mutagenesis PCR for point mutation in PvuII-site of K3 and K5.

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1,5 µl Primer 13 (10 pmol/µl)   95 30 sec
1,5 µl Primer 14 (10 pmol/µl)   58 45 sec x19
1 µl Template   72 4 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
ad 50 µl bidest. H2O  
PCR
Investigator: Christian
Aim: Construction of BioBricks → Deletion of restriction sites in iBB2 via mutagenesis PCR.

Mutagenesis PCR for point mutation in EcoRI-site of K3 and K5.

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1,5 µl Primer 15 (10 pmol/µl)   95 30 sec
1,5 µl Primer 16 (10 pmol/µl)   63 45 sec x19
1 µl Template   72 4 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
ad 50 µl bidest. H2O  

05.04.2013

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation of mutagenised pJet1.2 iBB2 into E. coli.

Transformation of products of the second PCR into E. coli DH5α ( 30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

08.04.2013

PCR
Investigator: Christian
Aim: Construction of BioBricks → Repeat of mutagenesis of pJet1.2 iBB2 and adding of pre- and suffix of iBB1, iBB6, iBB7 and iBB8.

PCR of iBB1 (pre- and suffix), iBB6 (pre- and suffix), and iBB2 (mutagenesis of PvuII-site), second PCR of iBB2 (mutagenesis of EcoRI-site), iBB7 (pre- and suffix) and iBB8 (pre- and suffix). All done with analog mix and program. Primers vary.

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw   95 30 sec
1 µl Primer rv   65 45 sec x19
1 µl Template   72 4 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
10 µl bidest. H2O  
Digest
Investigator: Christian
Aim: Preparation of pSB1C3 → Digestion of pSB1C3 J04450.

Digestion of pSB1C3 J04450 (2012) with EcoRI and PstI.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   pPha-NR cut with EcoRI and PstI.   2868 bp + 992 bp
3   pPha-T1 cut with EcoRI and PstI.   3554 bp + 541 bp

Lanes 2 and 3 contained bands at expected height.

Samples 2 and 3 are successful.

09.04.2013

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation of mutagenised pJet1.2 iBB2 into E. coli.

Transformation of the PCR-product of iBB2 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 45 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

Sequencing
Investigator: Christian
Aim: Construction of BioBricks → Sequencing results of iBB3 and iBB4.

IBB3 and iBB4: point mutations correct.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Adding of pre- and suffix via PCR.

PCR with iBB3 and iBB4 (pre- and suffix).

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw   95 30 sec
1 µl Primer rv   58 45 sec x19
1 µl Template   72 4 min
1 µl dNTPs   72 6 min
10 µl 5x buffer   4 Hold
35 µl bidest. H2O  

PCR
Investigator: Christian
Aim: Construction of BioBricks

The relevant fragment is cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   Mutagenesis of iBB2   717 bp
3   Mutagenesis of iBB7   768 bp
4   Mutagenesis of iBB8   1561 bp
5   Digest of pSB1C3-J04450   2000 bp

Band 3 shows a band at the expected heigt, band 4 lies to low, whereas the other two samples are hard to evaluate.

Mutagenesis of iBB7 (band 3) is positive.
PCR
Investigator: Christian
Aim: Construction of BioBricks → Repeat of adding pre- and suffix of iBB1 via PCR.

PCR of iBB1 repeated like 08.04.13.

Transformation
Investigator: Christian
Aim: Preparation of pSB1C3 → Transformation of pSB1C3 J04450 into E. coli.

Transformation of pSB1C3 J04450 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LBCM, overnight at 37°C.

10.04.2013

PCR
Investigator: Christian
Aim: Construction of BioBricks → Cleaning of PCR-products of iBB4.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Inoculation
Investigator: Christian
Aim: Construction of BioBricks and preparation of pSB1C3 → Inoculation of transformants.

Some colonies of pJet1.2 iBB2 inoculated in 5 ml LBamp and pSB1C3 J04450 inoculated in 5 ml LBCM, overnight at 37°C.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Adding of pre- and suffix of iBB5 via PCR.
Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1,5 µl Primer 15 (10 pmol/µl)   95 30 sec
1,5 µl Primer 16 (10 pmol/µl)   58 45 sec x30
1 µl Template (pPha-NR)   72 4 min
1 µl dNTPs   72 6 min
10 µl 5x buffer   4 Hold
35 µl bidest. H2O  

11.04.2013

Miniprep
Investigator: Christian
Aim: Construction of BioBricks and preparation of pSB1C3, → Miniprep of transformants.

Miniprep of pJet1.2 iBB2 and pSB1C3 J04450 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest
Investigator: Christian
Aim: Preparation of BioBrick-templates → Control digestion of pJet1.2 iBB2.
  • 3 µl DNA template
  • 1 µl EcoRI
  • 1 µl PstI
  • 2 µl buffer O (Fermentas)
  • 13 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

Expectations

  • pJet1.2: 2430 bp
  • iBB2: 740 bp

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-7   pJet1.2 + iBB2   2430 + 740 bp

We receive all expected fragments.

All positive.
Digest
Investigator: Christian
Aim: Preparation of pSB1C3 → Digestion with EcoRI and PstI.
  • 50 µl DNA template
  • 2 µl EcoRI
  • 4 µl PstI
  • 8 µl buffer O
  • 6 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

12.04.2013

Digest
Investigator: Christian
Aim: Construction of BioBricks → Digestion with EcoRI and PstI.

Digestion of iBB1, iBB2, iBB4, iBB5, iBB7 and iBB8.

  • 30 µl DNA template
  • 1 µl EcoRI
  • 1 µl PstI
  • 3,5 µl buffer O

The samples were incubated for 2 h at 37 °C.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Adding of pre- and suffix of iBB3 and iBB6 via PCR.
Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw   95 30 sec
1 µl Primer rv   58 45 sec x30
1 µl Template   72 4 min
1 µl dNTPs   72 6 min
10 µl buffer   4 Hold
35 µl bidest. H2O  

16.04.2013

Ligation
Investigator: Christian
Aim: Preparation of pSB1C3 → Testligation.

Dephosphorylation of pSB1C3 and testligation with iBB5.

Transformation
Investigator: Christian
Aim: Preparation of pSB1C3 → Transformation of pSB1C3 iBB5 into E. coli.

Transformation of pSB1C3 iBB5 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LBCM, overnight at 37°C.

Digest
Investigator: Christian
Aim: Construction of BioBricks → Digestion with EcoRI and PstI.

Digestion of iBB3 and iBB6.

Inoculation
Investigator: Christian
Aim: Preparation of pSB1C3 → Inoculation of pSB1C3 J04450 for miniprep.

Inoculation of pSB1C3 J04450 in LBCM, overnight at 37°C.

17.04.2013

Miniprep
Investigator: Christian
Aim: Preparation of pSB1C3 → Miniprep of pSB1C3 J04450.

Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest
Investigator: Christian
Aim: Preparation of pSB1C3 → Digestion of pSB1C3 J04450 with EcoRI and PstI.
  • 29 µl DNA template
  • 1 µl EcoRI
  • 1 µl PstI
  • 4 µl buffer O
  • 3 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content
2-3   pSB1C3-J04450 cut with EcoRI + PstI.

The plasmid is cut successfully.

Both positive.
Ligation
Investigator: Christian
Aim: Construction of BioBricks → Ligation of single BioBricks into pSB1C3.

Ligation of iBB1, iBB3, iBB4, iBB5, iBB6, iBB7 and iBB8 into pSB1C3.

18.04.2013

Ligation
Investigator: Christian
Aim: Preparation of pSB1C3 → Self-ligation test.

Ligation of empty pSB1C3.

19.04.2013

PCR
Investigator: Christian
Aim: Construction of BioBricks → Repeat of adding pre- and suffix of iBB8 via PCR.

Reason: Transformation of iBB8 failed, PCR of iBB8 repeated.

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw   95 30 sec
1 µl Primer rv   65 45 sec x19
1 µl Template   72 4 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
10 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
3-6   iBB8   1404 bp

We receive all expected fragments. The expected fragment in lane 2 is missing.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

22.04.2013

Miniprep
Investigator: Christian
Aim: Construction of BioBricks → Miniprep of single bricks in pSB1C3.

Miniprep of iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest
Investigator: Christian
Aim: Construction of BioBricks → Control digestion of single BioBricks in pSB1C3

Control digestion of iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7.

  • 3 µl DNA template
  • 1 µl EcoRI
  • 1 µl PstI
  • 2 µl buffer O (Fermentas)
  • 13 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   pSB1C3-iBB1 cut with EcoRI + PstI   375 bp + 2000 bp
3   pSB1C3-iBB3 cut with EcoRI + PstI   193 bp + 2000 bp
4   pSB1C3-iBB4 cut with EcoRI + PstI   422 + 2000 bp
5   pSB1C3-iBB5 cut with EcoRI + PstI   241 + 2000 bp
6   pSB1C3-iBB6 cut with EcoRI + PstI   273 + 2000 bp
7   pSB1C3-iBB7 cut with EcoRI + PstI   711 + 2000 bp

We receive expected fragments in samples 3 and 4.

2 out of 6 are successful.
Digest
Investigator: Christian
Aim: Construction of BioBricks → Digestion of iBB2 and iBB8.
  • 29 µl DNA template
  • 1 µl EcoRI
  • 1 µl PstI
  • 4 µl buffer O
  • 3 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-3   pSB1C3-iBB2   660 bp
4-7   pSB1C3-iBB8   1404 bp

We receive expected fragments for sample 2 and 3.

2 out of 6 are positive.
Ligation
Investigator: Christian
Aim: Construction of BioBricks → Ligation of iBB2 and iBB8 into pSB1C3.

Ligation of iBB2 and iBB8 into pSB1C3, overnight at 16 °C.

23.04.2013

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation pSB1C3 iBB2 and pSB1C3 iBB8 into E. coli.

Transformation of pSB1C3 iBB2 and pSB1C3 iBB8 into E. coli DH5α (30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB), plated on LBCM, overnight at 37°C.

Sequencing
Investigator: Christian
Aim: Construction of BioBricks → Sequencing.

PSB1C3 iBB1, iBB3, iBB4, iBB5, iBB6 and iBB7 sent for sequencing.

25.04.2013

Inoculation
Investigator: Christian
Aim: Construction of BioBricks → Inoculation of pSB1C3 iBB2 and pSB1C3 iBB8 for miniprep.

2 colonies of pSB1C3 iBB2 and iBB8 inoculated in 5 ml LBCM, overnight at 37 °C.

26.04.2013

Sequencing
Investigator: Franzi
Aim: Construction of BioBricks → Examination of sequencing results (23.4.13).

1C3 iBB1 K1 + K2 correct

1C3 iBB4 K1 + K2 correct

1C3 iBB7 K3 + K4 correct

1C3 iBB5 K1 + K2 Mutagenesis of XhoI-Site failed, only necessary for iGEM-standard 10 → unnecessary, rest correct

1C3 iBB3 K1 + K2 same point-Mutation → template?

1C3 iBB6 K1 + K2 same point-mutation → template?

Sequencing
Investigator: Franzi
Aim: Construction of BioBricks → Sequencing of pPhaNR.

Original pPhaNR was sent to sequencing using primers iMR25/26 for iBB3 and iBB6.

Miniprep
Investigator: Franzi
Aim: Construction of BioBricks → Miniprep.

PSB1C3 iBB8 K1 and pSB1C3 iBB2 K1-4 were isolated via Miniprep (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 40 l Elutionbuffer.

Digest
Investigator: Franzi
Aim: Construction of BioBricks → Control digestion of pSB1C3 iBB8 and iBB2.
  • 1 µl DNA template
  • 0,5 µl EcoRI
  • 0,5 µl PstI
  • 1 µl Cut Smart buffer
  • 7 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   pSB1C3-iBB8   1404 bp
3-6   pSB1C3-iBB2   660 bp

IBB8 negative, iBB2 all positive.

Sequencing
Investigator: Franzi
Aim: Construction of BioBricks → Sequencing of pSB1C3 iBB2.

IBB2 K2 + K4 sequenced with primer VF2_fwd and VR_rev.

29.04.2013

PCR
Investigator: Franzi
Aim: Construction of BioBricks → Adding of pre- and suffix of iBB8 via PCR.
Volume Reagent   Temp (°C) Time
1 µl Phusion-Polymerase   95 3 min
1 µl Primer 33 fw   95 30 sec
1 µl Primer 34 rv   65 40 sec x30
1 µl 2xNR template   72 1 min
1 µl dNTPs   72 7 min
2 µl DMSO   4 Hold
10 µl 5xGC buffer  
33 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-3   iBB8, PCR I   1404 bp
5-6   iBB8, PCR II   1404 bp

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl bidest. H2O.

All positive.

Digest
Investigator: Franzi
Aim: Construction of BioBricks → Digestion of iBB8
  • 30 µl DNA template
  • 1 µl EcoRI-HF
  • 1 µl PstI-HF
  • 3,6 µl Cut Smart buffer

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
Ligation
Investigator: Franzi
Aim: Contruction of BioBricks → Assemble the Biobrick iBB8 into the vector pSB1C3.
  • 30 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 270 ng insert DNA (iBB8)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated overnight at 16 °C.

30.04.2013

Transformation
Investigator: Franzi
Aim: Contruction of BioBricks → Transformation of the plasmid pSB1C3 iBB8 in E. coli.

The complete ligation sample was mixed with one aliquot of chemo-competent E. coli DH5α cells (50 µl) and incubated for 30 min on ice. The heatshock was performed at 42 °C for 60 sec and cells were then incubated at 37 °C for 1 h with 900 µl LB. The sample was concentrated and plated on LB containing chloramphenicol.
The plate was incubated for two days at 28 °C .

Transformation
Investigator: Franzi
Aim: Contruction of BioBricks → Transformation of 2xNR HC+LC.

2xNR HC + LC retransformed as transcribed above. Plated on LB containing ampicillin.

Sequencing
Investigator: Franzi
Aim: Construction of BioBricks → sequencing results.

Sequencing of iBB2 K2 + K4 failed (possibly secondary structure).

Sequenced again with primer cat_int_rev.

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