Team:Goettingen/NoteBook w5

From 2013.igem.org

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<div class="monat">July</div>
 
 +
<div class="tlob start" id="tl_0705">
 +
<span class="date">05th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-rt">Colonies on LB<sup>Amp</sup> plates!!!</p>
 +
<div class="timeline-cont">
 +
<p class="MsoNormal"><span lang="EN-US">Transformation of Ligation with
 +
GFP-Terminator insert and part 8 backbone worked!!!</span></p>
-
<div class="tlob start" id="tl_0604">
+
<p class="MsoNormal"><span lang="EN-US">No clones on negative control, but very few
 +
clones on without insert control. </span><span lang="EN-US" style="font-family:
 +
Wingdings">à</span><span lang="EN-US"> possibly again incomplete digestion of
 +
part 8 (just 20 min) </span><span lang="EN-US" style="font-family:Wingdings">à</span><span lang="EN-US"> use 1 h for dig, since reaction for gel ex yesterday seemed to be
 +
more completely digested…. </span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Stored at 4 °C</span></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">No colonies on LB<sup>Cm</sup> plates</span></b></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;">
 +
</span></span><span lang="EN-US">Further incubation till next morning</span></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
</div>
 +
</div>
 +
<br />
 +
 
 +
<div class="tlob start" id="tl_0704">
 +
<span class="date">04th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-rt">Rerstriction digestion of part 7 and the GFP-Terminator construct</p>
 +
<div class="timeline-cont">
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Part 7:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">15 µl part 7 plasmid DNA (1 µg)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">10 µl NEB buffer 2.1</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">2 µl EcoRI</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">2 µl PstI</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">71 µl H<sub>2</sub>O</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">GFP-Term:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">5 µl GFP-Term C1 plasmid DNA (1 µg)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">10 µl NEB buffer 2.1</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">2 µl XbaI</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">2 µl PstI</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">81 µl H<sub>2</sub>O</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Both reactions were split into 2x 50µl (for
 +
more thorough reaction)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Both incubated for 30 min at 37°C, then
 +
heat inactivation for 20 min at 80°C</span></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Gel-Extraction of pSB1C3 from the
 +
restriction digest of part 7 (EcoRI+PstI) for future ligations</span></b></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Gel</span></b><span lang="EN-US">:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><img width="408" id="图片 2" src="https://static.igem.org/mediawiki/2013/9/9e/Goe-04.07.13-RT-1.png" alt="am geldoc 2013-07-04 14hr 41min"></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Purification of DNA from gel using
 +
Qiagen PCR purification kit:</span></b></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span><span lang="EN-US">like Katrin 25.06.2013</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Elution with 30 µl HPLC water (pre-warmed)</span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">NanoDrop:</span></b></p>
 +
 
 +
<table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" align="left" style="border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:
 +
4.8pt">
 +
<tbody><tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">pSB1C3 clone</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border:solid windowtext 1.0pt;
 +
  border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">Concentration (ng/µl)</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border:solid windowtext 1.0pt;
 +
  border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">A<sub>260nm</sub>/A<sub>280nm</sub></span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border:solid windowtext 1.0pt;
 +
  border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">A<sub>260nm</sub>/A<sub>230nm</sub></span></p>
 +
  </td>
 +
</tr>
 +
<tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">6.2</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border-top:none;border-left:none;
 +
  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1.35</span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">0.12</span></p>
 +
  </td>
 +
</tr>
 +
<tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">2</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">11.3</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border-top:none;border-left:none;
 +
  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1.27</span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">0.21</span></p>
 +
  </td>
 +
</tr>
 +
<tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">3</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">10.2</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border-top:none;border-left:none;
 +
  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1.71</span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">0.06</span></p>
 +
  </td>
 +
</tr>
 +
</tbody></table>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">&nbsp;</span></b></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">&nbsp;</span></b></p>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;">
 +
</span></span><span lang="EN-US">DNA frozen away in one of the red boxes with
 +
today´s date on the vial.</span></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Clonation of the RBS-GFP-Terminator
 +
Construct:</span></b></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Gel-Extraction of the GFP-Terminator
 +
construct from the restriction digest (XbaI+PstI), ligation into opened
 +
RBS-Vector (Part 8) and transformation</span></b></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Gel:</span></b></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><img width="429" id="图片 1" src="https://static.igem.org/mediawiki/2013/5/5d/Goe-04.07.13-RT-2.png" alt="am geldoc 2013-07-04 14hr 39min"></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Purification of DNA from gel using
 +
Qiagen PCR purification kit:</span></b></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span><span lang="EN-US">like Katrin 25.06.2013</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span><span lang="EN-US">Elution with 30 µl HPLC water (pre-warmed)</span></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">NanoDrop:</span></b></p>
 +
 
 +
<table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" align="left" style="border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:
 +
4.8pt">
 +
<tbody><tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">GFP-Term clone</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border:solid windowtext 1.0pt;
 +
  border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">Concentration (ng/µl)</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border:solid windowtext 1.0pt;
 +
  border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">A<sub>260nm</sub>/A<sub>280nm</sub></span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border:solid windowtext 1.0pt;
 +
  border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">A<sub>260nm</sub>/A<sub>230nm</sub></span></p>
 +
  </td>
 +
</tr>
 +
<tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">5.4</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border-top:none;border-left:none;
 +
  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1.75</span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">0.07</span></p>
 +
  </td>
 +
</tr>
 +
<tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">2</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">19.5</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border-top:none;border-left:none;
 +
  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1.62</span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">0.19</span></p>
 +
  </td>
 +
</tr>
 +
<tr>
 +
  <td width="109" valign="top" style="width:81.7pt;border:solid windowtext 1.0pt;
 +
  border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">3</span></p>
 +
  </td>
 +
  <td width="128" valign="top" style="width:95.95pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">6.2</span></p>
 +
  </td>
 +
  <td width="89" valign="top" style="width:66.9pt;border-top:none;border-left:none;
 +
  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">1.32</span></p>
 +
  </td>
 +
  <td width="79" valign="top" style="width:59.55pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:9.0pt">0.10</span></p>
 +
  </td>
 +
</tr>
 +
</tbody></table>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">&nbsp;</span></b></p>
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">&nbsp;</span></b></p>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;">
 +
</span></span><span lang="EN-US">DNA frozen away in one of the red boxes with
 +
today´s date on the vial.</span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Ligation</span></b></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-Clone 2 was used for ligation to the
 +
opened RBS-Vector (also known as part 8 –see 1.7.13)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-ligation was pipetted as follows:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">13.5µl HPLC H2O</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">2µl Ligase buffer</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">2.5µl RBS-Vector DNA (~20ng)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">1µl GFP-Term Clone 2 DNA (~20ng)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">1µl Ligase</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-For the control, the GFP-Term.-Insert was
 +
substituted with HPLC H2O</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-Ligation was performed at RT for 10
 +
minutes</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-Heat-inactivation was performed at 80°C
 +
for 20 minutes</span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b><span lang="EN-US">Transformation</span></b></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-The whole ligation was used for the
 +
transformation.</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">-Transformation performed according to the
 +
methods-folder</span></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Extraction of Terminator and RBS from cultures prepared on 03.07.<br />Digestion of extracted plasmids containing Terminator and RBS</p>
 +
<div class="timeline-cont">
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Extraction of Terminator and RBS from cultures prepared
 +
  on 03.07.</span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">extraction
 +
  of plasmids via NucleoSpin<sup>R</sup>Plasmid kit</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">concentration
 +
  measurement on Nanodrop</span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="margin-left:36.7pt;border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Sample</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Concentration</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">T1</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">69 ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">T2</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">80 ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">T3</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">118 ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">T4</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">95 ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">RBS1</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">too low</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">RBS2</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">52 ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">RBS1</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">too low</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="250" valign="top" style="width:187.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">RBS1</span></p>
 +
    </td>
 +
    <td width="230" valign="top" style="width:172.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">too low</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Digestion of extracted plasmids containing Terminator
 +
  and RBS </span></b></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">followed protocol
 +
  for digestion as written in BioBrick<sup>R</sup>Assembly Kit</span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="margin-left:50.9pt;border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="296" valign="top" style="width:222.15pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Terminator:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></p>
 +
    </td>
 +
    <td width="232" valign="top" style="width:173.85pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="296" valign="top" style="width:222.15pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plasmid
 +
    DNA (pSB1C3)</span></p>
 +
    </td>
 +
    <td width="232" valign="top" style="width:173.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">500ng</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="296" valign="top" style="width:222.15pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">PstI</span></p>
 +
    </td>
 +
    <td width="232" valign="top" style="width:173.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="296" valign="top" style="width:222.15pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">EcoRI</span></p>
 +
    </td>
 +
    <td width="232" valign="top" style="width:173.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="296" valign="top" style="width:222.15pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">NEB
 +
    cutter buffer (10x)</span></p>
 +
    </td>
 +
    <td width="232" valign="top" style="width:173.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="296" valign="top" style="width:222.15pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
 +
    </td>
 +
    <td width="232" valign="top" style="width:173.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">to
 +
    50µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for 10min at 37°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">heat inactivation for 20min at 80°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">store at -20°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <table class="MsoTableGrid" border="0" cellspacing="0" cellpadding="0" style="margin-left:54.0pt;border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="156" valign="top" style="width:117.35pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:
 +
    0cm;margin-bottom:0cm;margin-left:-1.45pt;margin-bottom:.0001pt;line-height:
 +
    normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">M| T1| T2|
 +
    T3| T4</span></p>
 +
    <p class="MsoListParagraphCxSpLast" style="margin:0cm;margin-bottom:.0001pt;
 +
    line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;"><img border="0" width="121" height="168" id="图片 7" src="https://static.igem.org/mediawiki/2013/c/cb/Goe-dac-Image016.gif" alt="20130704_terminatorPart7_digest"></span></p>
 +
    </td>
 +
    <td width="342" valign="top" style="width:256.25pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
    text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings;color:#00B050">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;color:#00B050">Digestion
 +
    failed</span></p>
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
    text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">set up digestion
 +
    again</span></p>
 +
    <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
    text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">add 1µl of PstI
 +
    and 1µl of EcoRI-HF</span></p>
 +
    <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
    text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for
 +
    45min at 37°C</span></p>
 +
    <p class="MsoListParagraph" style="margin:0cm;margin-bottom:.0001pt;
 +
    line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">RBS:</span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="margin-left:43.8pt;border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="269" valign="top" style="width:201.4pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" align="center" style="margin-top:0cm;
 +
    margin-right:0cm;margin-bottom:0cm;margin-left:8.75pt;margin-bottom:.0001pt;
 +
    text-align:center;line-height:normal"><span lang="EN-US" style="font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">Components</span></p>
 +
    </td>
 +
    <td width="211" valign="top" style="width:158.6pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" align="center" style="margin-bottom:0cm;
 +
    margin-bottom:.0001pt;text-align:center;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="269" valign="top" style="width:201.4pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
    margin-bottom:0cm;margin-left:8.75pt;margin-bottom:.0001pt;line-height:
 +
    normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plasmid
 +
    DNA (pSB1C3)</span></p>
 +
    </td>
 +
    <td width="211" valign="top" style="width:158.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">10µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="269" valign="top" style="width:201.4pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
    margin-bottom:0cm;margin-left:8.75pt;margin-bottom:.0001pt;line-height:
 +
    normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">PstI</span></p>
 +
    </td>
 +
    <td width="211" valign="top" style="width:158.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="269" valign="top" style="width:201.4pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
    margin-bottom:0cm;margin-left:8.75pt;margin-bottom:.0001pt;line-height:
 +
    normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">EcoRI</span></p>
 +
    </td>
 +
    <td width="211" valign="top" style="width:158.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="269" valign="top" style="width:201.4pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
    margin-bottom:0cm;margin-left:8.75pt;margin-bottom:.0001pt;line-height:
 +
    normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">NEB
 +
    cutter buffer (10x)</span></p>
 +
    </td>
 +
    <td width="211" valign="top" style="width:158.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="269" valign="top" style="width:201.4pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
    margin-bottom:0cm;margin-left:8.75pt;margin-bottom:.0001pt;line-height:
 +
    normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
 +
    </td>
 +
    <td width="211" valign="top" style="width:158.6pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">33µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoListParagraphCxSpFirst" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for 30min at 37°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">heat inactivation for 20min at 80°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">store at -20°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">M |R2 |T1 |T2| T3| T4</span></p>
 +
  <p class="MsoListParagraphCxSpLast" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;line-height:normal"><span lang="EN-US"><img border="0" width="159" height="187" id="图片 10" src="https://static.igem.org/mediawiki/2013/a/ab/Goe-dac-Image017.gif" alt="20130705_digestion_Terminator_RBS"></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
 
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
</div>
 +
</div>
 +
<br />
 +
 
 +
<div class="tlob start" id="tl_0703">
 +
<span class="date">03th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 +
<div class="cont">
 +
<p class="timeline-title goe-rt">Restriction
 +
Digest of Parts 1-4 for cloning from 1.7.13 - Run on 4% gel</p>
 +
<div class="timeline-cont">
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">0.5 µl uncut plasmid + 3.5 µl
 +
dH<sub>2</sub>O + 1 µl LD 5x</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">Ca. 40 ng of R.D. of plasmids
 +
(= 4 µl) supplied with 1 µl of LD 5x</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">Run at 100 V on 4
 +
%-agarose-1xTAE gel; marker: log ladder (3 µl)</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">EtBr staining, destaining, UV
 +
detection</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Uncut plasmids visible (between 3 and 8kb,
 +
although about 2.5kb big), linearized plasmids visible (between 8 and 10 kb…),
 +
promoter fragments not visible at all (should be 30-120bps). Will add picture
 +
later. <b style="mso-bidi-font-weight:normal"><o:p></o:p></b></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Discussion:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">The thick gel might have unrepresentative
 +
effects on large fragments (e.g. plasmids). This thesis is supported by the
 +
unreadable marker bands between 3 and 8 kb.</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">The promoter fragments might just be too
 +
small to be stained properly or they are not in the constructs. Only sequencing
 +
can solve this riddle…</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Wells used as follows:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">M_Part1uncut_Part1RD_Part2uncut_Part2RD_Part3uncut_Part3RD_Part4uncut_part4RD_M<b style="mso-bidi-font-weight:normal"><o:p></o:p></b></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">used Marker: NEB Quick-Load 2-Log DNA
 +
Ladder (0.1-10kb)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shapetype
 +
id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t"
 +
path="m@4@5l@4@11@9@11@9@5xe" filled="f" stroked="f">
 +
<v:stroke joinstyle="miter"/>
 +
<v:formulas>
 +
  <v:f eqn="if lineDrawn pixelLineWidth 0"/>
 +
  <v:f eqn="sum @0 1 0"/>
 +
  <v:f eqn="sum 0 0 @1"/>
 +
  <v:f eqn="prod @2 1 2"/>
 +
  <v:f eqn="prod @3 21600 pixelWidth"/>
 +
  <v:f eqn="prod @3 21600 pixelHeight"/>
 +
  <v:f eqn="sum @0 0 1"/>
 +
  <v:f eqn="prod @6 1 2"/>
 +
  <v:f eqn="prod @7 21600 pixelWidth"/>
 +
  <v:f eqn="sum @8 21600 0"/>
 +
  <v:f eqn="prod @7 21600 pixelHeight"/>
 +
  <v:f eqn="sum @10 21600 0"/>
 +
</v:formulas>
 +
<v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
 +
<o:lock v:ext="edit" aspectratio="t"/>
 +
</v:shapetype><v:shape id="图片_x0020_18" o:spid="_x0000_i1027" type="#_x0000_t75"
 +
alt="am geldoc 2013-07-03 13hr 40min" style='width:226.5pt;height:169.5pt;
 +
visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Restriction%20Digest%20of%20Parts%201.files/image001.png"
 +
  o:title="am geldoc 2013-07-03 13hr 40min"/>
 +
</v:shape><![endif]--><!--[if !vml]--><img width="302" height="226" src="https://static.igem.org/mediawiki/2013/c/ca/Goe-03.07.13-RT-1.png" alt="am geldoc 2013-07-03 13hr 40min" v:shapes="图片_x0020_18"><!--[endif]--></span></p>
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Restriction
 +
digestion of isolated plasmids from yesterday run on 1% gel (for 1.5h)<o:p></o:p></span></b></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">can not open scn file. Saved under Reporter
 +
team gel doc in folder with this date (gel doc 2013-07-03 15hr 29min, control =
 +
14hr 34 min)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span>Wells as following:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">RiboA (C1,C2,C3), RiboB (C1, C2, C3), RiboC
 +
(C1), RiboD (C1, C2, C3), lin part7 (C1, C2, C3), DarR (C1, C2, C4), GFP
 +
(C1,C2,C4), pSB1C3 (C1, C2, C3)</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">same order of wells for control</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Control:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shape
 +
id="图片_x0020_16" o:spid="_x0000_i1026" type="#_x0000_t75" alt="am geldoc 2013-07-03 15hr 34min"
 +
style='width:318pt;height:237.75pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Restriction%20Digest%20of%20Parts%201.files/image003.png"
 +
  o:title="am geldoc 2013-07-03 15hr 34min"/>
 +
</v:shape><![endif]--><!--[if !vml]--><img width="424" src="https://static.igem.org/mediawiki/2013/b/b4/Goe-03.07.13-RT-2.png" alt="am geldoc 2013-07-03 15hr 34min" v:shapes="图片_x0020_16"><!--[endif]--></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Digested:</span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shape
 +
id="图片_x0020_17" o:spid="_x0000_i1025" type="#_x0000_t75" alt="am geldoc 2013-07-03 15hr 29min"
 +
style='width:318pt;height:237.75pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Restriction%20Digest%20of%20Parts%201.files/image005.png"
 +
  o:title="am geldoc 2013-07-03 15hr 29min"/>
 +
</v:shape><![endif]--><!--[if !vml]--><img width="424" src="https://static.igem.org/mediawiki/2013/f/f7/Goe-03.07.13-RT-3.png" alt="am geldoc 2013-07-03 15hr 29min" v:shapes="图片_x0020_17"><!--[endif]--></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">GFP-Terminator construct could be cloned successfully (fragment of
 +
slightly &gt; 1 kb corresponding likely to GFP (ca. 900 bp) and terminator (ca.
 +
170 bp) visible)</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">Clones derived from transformation of linearzised part 7 contain
 +
apparently plasmid with part 7 (terminator) </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> ca. 200 bp fragment visible after digest, corresponding presumably
 +
to terminator </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
 +
&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
 +
mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:
 +
Wingdings">à</span></span><span lang="EN-US"> clones result possibly from
 +
transformation of not linearized plasmids present after incomplete digest of
 +
part 7</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">For C2 of RiboB cloning, a weak band at ca. 200 bp could be
 +
detected, that does not fit with the expected insert of 370 bp </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> something else got cloned…</span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">For transformations of ligation reactions containing linearized
 +
pSB1C3 as a vector, no transformants containing the desired inserts could be
 +
obtained </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
 +
&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
 +
mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:
 +
Wingdings">à</span></span><span lang="EN-US"> plasmid that was transformed could
 +
be one of the residual template plasmids containing Promoter-RBS-RFP-Terminator
 +
(ca. 1.5 kb): after R.D., bands at 1.5 kb and 2 kb corresponding to the
 +
expected fragments could be detected. However, band at 1.5 kb is very weak, and
 +
for uncut plasmids, a strong band at this height representing the supercoiled
 +
plasmid could be detected </span><span lang="EN-US" style="font-family:Wingdings;
 +
mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
 +
mso-char-type:symbol;mso-symbol-font-family:Wingdings"><span style="mso-char-type:
 +
symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> it’s
 +
also possible that pSB1C3 can’t be restricted properly… anyway, linearized
 +
pSB1C3 seems not to work for cloning </span><span lang="EN-US" style="font-family:
 +
Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
 +
mso-char-type:symbol;mso-symbol-font-family:Wingdings"><span style="mso-char-type:
 +
symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> cut
 +
out terminator from part 7, extract backbone (= linearized pSB1C3) and use it
 +
for cloning of Ribo- and DarR biobricks instead… </span></p>
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Sequencing
 +
data arrived!<o:p></o:p></span></b></p>
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;
 +
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
 +
Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US">No biobrick in the DarR operator construct!<o:p></o:p></span></b></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Transformation of T7 promoter<br />Cultivation of E.coli containing Terminator and RBS</p>
 +
<div class="timeline-cont">
 +
<td width="643" valign="top" style="width:17.0cm;border-top:none;border-left:
 +
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt">
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Transformation of T7 promoter</span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></b></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">use T7
 +
  promoter BBa_1712074 in pSB1AK8 backbone (plate 5, 6N)</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">resuspend
 +
  in 10µl H<sub>2</sub>O</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation
 +
  at RT for 5min.</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">transformation
 +
  of 2µl T7 promoter with 200µl of comp. E.coli cells and incubation for 30min
 +
  on ice</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">90sec
 +
  heat shock at 42°C</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5min on
 +
  ice</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">add
 +
  800µl LB medium and incubate for 1h at 37°C with agitation</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plate
 +
  out 100µl of transformation mixture on LB medium containing amikacin </span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">centrifuge
 +
  tubes for 1min, discard 800µl and plate out the rest on new plates</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubate
 +
  plates for 2 days at 37°C</span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Cultivation of E.coli containing Terminator and RBS </span></b></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">reporter
 +
  team cloned terminator (BBa_B0015; pSB1C3 backbone; Cm-resistance) and RBS
 +
  (BBa_0034; pBS1A2; Amp-resistance) into E.coli</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">inoculation
 +
  of each 4 liquid cultures in LB containing Cm and Amp</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation
 +
  at 37°C o/n with agitation</span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  </td>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
</div>
 +
</div>
 +
<br />
 +
 
 +
<div class="tlob start" id="tl_0702">
 +
<span class="date" style="left:-5px">02nd<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
 +
<div class="cont" style="left:-10px">
 +
<p class="timeline-title goe-rt">Plasmid Mini Preparation</p>
 +
<div class="timeline-cont">
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Back-up
 +
plates<o:p></o:p></span></b></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">for all inoculated clones
 +
(except for clones containing plasmids for parts 1 – 4)<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">incubation over day at 37 °C<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">stored at 4 °C<o:p></o:p></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Plasmid
 +
Mini Preparation<o:p></o:p></span></b></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">for all inoculated clones<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">kit from Macherey-Nagel<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">elution with pre-warmed 30 µl
 +
HPLC water<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">DarR biobrick clone 3: almost
 +
all lysate spilled…<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-fareast-font-family:&quot;Times New Roman&quot;"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><!--[endif]--><span lang="EN-US">Concentration measurement on
 +
NanoDrop<b style="mso-bidi-font-weight:normal"><o:p></o:p></b></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Concentrations:<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shapetype id="_x0000_t75"
 +
coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe"
 +
filled="f" stroked="f">
 +
<v:stroke joinstyle="miter"/>
 +
<v:formulas>
 +
  <v:f eqn="if lineDrawn pixelLineWidth 0"/>
 +
  <v:f eqn="sum @0 1 0"/>
 +
  <v:f eqn="sum 0 0 @1"/>
 +
  <v:f eqn="prod @2 1 2"/>
 +
  <v:f eqn="prod @3 21600 pixelWidth"/>
 +
  <v:f eqn="prod @3 21600 pixelHeight"/>
 +
  <v:f eqn="sum @0 0 1"/>
 +
  <v:f eqn="prod @6 1 2"/>
 +
  <v:f eqn="prod @7 21600 pixelWidth"/>
 +
  <v:f eqn="sum @8 21600 0"/>
 +
  <v:f eqn="prod @7 21600 pixelHeight"/>
 +
  <v:f eqn="sum @10 21600 0"/>
 +
</v:formulas>
 +
<v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
 +
<o:lock v:ext="edit" aspectratio="t"/>
 +
</v:shapetype><v:shape id="图片_x0020_1" o:spid="_x0000_i1025" type="#_x0000_t75"
 +
alt="concentrations_2" style='width:282.75pt;height:389.25pt;visibility:visible;
 +
mso-wrap-style:square'>
 +
<v:imagedata src="0702.files/image001.jpg" o:title="concentrations_2"/>
 +
</v:shape><![endif]--><!--[if !vml]--><img width="377" height="519" src="https://static.igem.org/mediawiki/2013/b/ba/Goe-02.07.13-RT.png" alt="concentrations_2" v:shapes="图片_x0020_1"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></b></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">DarR biobrick clone 3 has
 +
strangely high concentration<o:p></o:p></span></b></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Test
 +
restriction digest of isolated plasmids<o:p></o:p></span></b></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">Cut out inserts with EcoRI and PstI (Fermentas/ThermoScientific
 +
enzymes, not distribution kit!)<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">Only 3 clones tested, if there are 5 to waste less enzyme…<o:p></o:p></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">For
 +
1 reaction (</span></b><span lang="EN-US">10 µl total volume)<o:p></o:p></span></p>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">0.5 µl PstI<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">0.5 µl EcoRI<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">1 µl buffer 0 (both enzymes have 100 %
 +
activity and no star activity in this buffer)<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">3 µl plasmid DNA mix containing ca. 200 –
 +
300 ng plasmid (see picture of NanoDrop table above, rightmost + smallest part
 +
of table)<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">5 µl dH<sub>2</sub>O<o:p></o:p></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">Master Mix consisting of<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">12.5 µl PstI<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">12.5 µl EcoRI<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">25 µl buffer 0<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">125 µl dH<sub>2</sub>O<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">In total: 250 µl<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal" style="margin-left:54.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings;
 +
mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span></span><!--[endif]--><span lang="EN-US">Add 7 µl to each plasmid DNA mix<o:p></o:p></span></p>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Incubation for ca. 1 h at 37 °C<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US">Freezing of samples at -20°C<o:p></o:p></span></p>
 +
 
 +
<p class="MsoNormal"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
<p class="timeline-title goe-dac">Digestion of plasmid pSB1C3 with EcoRI and PstI from
 +
  linear precursor<br />Dephosphorylation of vector pSB1C3<br />Ligation of Strep-Tag; S. aureus DAC domain purified and S.aureus non purified with pSB1C3<br />Transformation of ligated samples with competent E.coli cells</p>
 +
<div class="timeline-cont">
 +
 
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Digestion of plasmid pSB1C3 with EcoRI and PstI from
 +
  linear precursor</span></b></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></b></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Components:</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plasmid
 +
    DNA (pSB1C3)</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">20µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">PstI</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">EcoRI</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">NEB
 +
    cutter buffer (10x)</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">23µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
  .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for 30min at 37°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">heat inactivation for 20min at 65°C</span></p>
 +
  <p class="MsoListParagraphCxSpMiddle" style="margin-top:0cm;margin-right:0cm;
 +
  margin-bottom:0cm;margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;
 +
  line-height:normal"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">store at -20°C</span></p>
 +
  <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
  .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Dephosphorylation of vector pSB1C3</span></b></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="295" valign="top" style="width:221.2pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Components:</span></p>
 +
    </td>
 +
    <td width="233" valign="top" style="width:174.8pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="295" valign="top" style="width:221.2pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">digested
 +
    pSB1C3</span></p>
 +
    </td>
 +
    <td width="233" valign="top" style="width:174.8pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">50µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="295" valign="top" style="width:221.2pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">antarch’s
 +
    buffer (10x)</span></p>
 +
    </td>
 +
    <td width="233" valign="top" style="width:174.8pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="295" valign="top" style="width:221.2pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">antarch’s
 +
    phosphatase</span></p>
 +
    </td>
 +
    <td width="233" valign="top" style="width:174.8pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoListParagraph" style="margin-left:0cm;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Measurement of DNA concentration</span></b></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="322" valign="top" style="width:241.65pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Sample</span></p>
 +
    </td>
 +
    <td width="206" valign="top" style="width:154.35pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Concentration</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="322" valign="top" style="width:241.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">pSB1C3 (digested/dephosphorylated)&nbsp;&nbsp; </span></p>
 +
    </td>
 +
    <td width="206" valign="top" style="width:154.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">5,4ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="322" valign="top" style="width:241.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">Strep-Tag
 +
    (digested)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
    </span></p>
 +
    </td>
 +
    <td width="206" valign="top" style="width:154.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">126ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="322" valign="top" style="width:241.65pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">S. areus DAC domain
 +
    (digested)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></p>
 +
    </td>
 +
    <td width="206" valign="top" style="width:154.35pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    &quot;Arial&quot;,&quot;sans-serif&quot;">181ng/µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Ligation of Strep-Tag; S. aureus DAC domain purified
 +
  and S.aureus non purified with pSB1C3</span></b></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Strep-Tag -
 +
    components:</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">insert
 +
    (Strep-Tag)</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plasmid
 +
    DNA (pSB1C3)</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">T4
 +
    ligase</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">T4
 +
    ligase buffer (10x)</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="292" valign="top" style="width:219.1pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
 +
    </td>
 +
    <td width="236" valign="top" style="width:176.9pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">14µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none">
 +
  <tbody><tr>
 +
    <td width="384" valign="top" style="width:288.25pt;border:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">S.aureus purified
 +
    and non purified - components:</span></p>
 +
    </td>
 +
    <td width="144" valign="top" style="width:107.75pt;border:solid black 1.0pt;
 +
    border-left:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">Volume</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="384" valign="top" style="width:288.25pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">insert
 +
    (S. aureus DAC domain)</span></p>
 +
    </td>
 +
    <td width="144" valign="top" style="width:107.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="384" valign="top" style="width:288.25pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plasmid
 +
    DNA (pSB1C3)</span></p>
 +
    </td>
 +
    <td width="144" valign="top" style="width:107.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="384" valign="top" style="width:288.25pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">T4
 +
    ligase</span></p>
 +
    </td>
 +
    <td width="144" valign="top" style="width:107.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">1µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="384" valign="top" style="width:288.25pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">T4
 +
    ligase buffer (10x)</span></p>
 +
    </td>
 +
    <td width="144" valign="top" style="width:107.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">2µl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td width="384" valign="top" style="width:288.25pt;border:solid black 1.0pt;
 +
    border-top:none;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpFirst" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">H<sub>2</sub>O</span></p>
 +
    </td>
 +
    <td width="144" valign="top" style="width:107.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
 +
    padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoListParagraphCxSpLast" style="margin-bottom:0cm;margin-bottom:
 +
    .0001pt;line-height:normal"><span lang="EN-US" style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">14µl</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubation for 1h
 +
  at room temperature</span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
  &quot;Arial&quot;,&quot;sans-serif&quot;">Transformation of ligated samples with competent E.coli
 +
  cells</span></b></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">add
 +
  ligation sample to 200µl of comp. cells and incubate for 30min on ice</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">each 2µl and 10µl
 +
  of ligated Strep-Tag, DAC domain S.aureus and relegation control used</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">90sec
 +
  heat shock at 42°C</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">5min on
 +
  ice</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">add
 +
  800µl LB medium and incubate for 1h at 37°C with agitation</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">plate
 +
  out 100µl of transformation mixture on LB medium containing 35µg/ml chloamph.
 +
  </span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">centrifuge
 +
  tubes for 1min, discard 800µl and plate out the rest on new plates</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">incubate
 +
  plates for 2 days at 37°C</span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
  <p class="MsoNormal" align="left" style="margin-left:54.0pt;text-align:left;
 +
  text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:Wingdings;color:#00B050">è<span style="font:7.0pt &quot;Times New Roman&quot;"> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;color:#00B050">Transformation
 +
  failed</span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;">&nbsp;</span></p>
 +
 
 +
 
 +
<div class="fbutton">Fold ↑</div>
 +
</div>
 +
</div>
 +
</div>
 +
<br />
 +
 
 +
<div class="tlob start" id="tl_0701">
<span class="date">01st<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<span class="date">01st<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
<div class="cont">
<div class="cont">
-
<p class="timeline-title">Restriction of parts 1 – 4 to cut out promoters for ligation with operator plasmid and restriction of part 8 for inserting GFP-Terminator cassette</p>
+
<p class="timeline-title goe-rt">Restriction of parts 1 – 4 to cut out promoters for ligation with operator plasmid and restriction of part 8 for inserting GFP-Terminator cassette</p>
<div class="timeline-cont">
<div class="timeline-cont">
-
<td width="1057" valign="top" style="width:792.9pt;border:solid windowtext 1.0pt;
+
 
-
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:943.5pt">
+
   <p class="MsoNormal"><span lang="EN-US">Reactions according to manual of
   <p class="MsoNormal"><span lang="EN-US">Reactions according to manual of
   Distribution kit:<o:p></o:p></span></p>
   Distribution kit:<o:p></o:p></span></p>
Line 233: Line 1,752:
   sample or the purification was not very efficient. Eluded a second time </span></p>
   sample or the purification was not very efficient. Eluded a second time </span></p>
   <p class="MsoNormal"><span lang="EN-US">with 20µl, no DNA found.<b style="mso-bidi-font-weight:normal"><o:p></o:p></b></span></p>
   <p class="MsoNormal"><span lang="EN-US">with 20µl, no DNA found.<b style="mso-bidi-font-weight:normal"><o:p></o:p></b></span></p>
-
  </td>
 
 +
<div class="fbutton">Fold ↑</div>
</div>
</div>
 +
<p class="timeline-title goe-dac">Primer iGEM_17 designed (used for deletion of the restriction site in DAC domain of S. pneumoniae</p>
 +
<div class="timeline-cont">
 +
 +
 +
<div class="fbutton">Fold ↑</div>
 +
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Latest revision as of 19:59, 30 September 2013

05th

Colonies on LBAmp plates!!!

Transformation of Ligation with GFP-Terminator insert and part 8 backbone worked!!!

No clones on negative control, but very few clones on without insert control. à possibly again incomplete digestion of part 8 (just 20 min) à use 1 h for dig, since reaction for gel ex yesterday seemed to be more completely digested….

Stored at 4 °C

No colonies on LBCm plates

è Further incubation till next morning

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04th

Rerstriction digestion of part 7 and the GFP-Terminator construct

Part 7:

15 µl part 7 plasmid DNA (1 µg)

10 µl NEB buffer 2.1

2 µl EcoRI

2 µl PstI

71 µl H2O

 

GFP-Term:

5 µl GFP-Term C1 plasmid DNA (1 µg)

10 µl NEB buffer 2.1

2 µl XbaI

2 µl PstI

81 µl H2O

Both reactions were split into 2x 50µl (for more thorough reaction)

Both incubated for 30 min at 37°C, then heat inactivation for 20 min at 80°C

Gel-Extraction of pSB1C3 from the restriction digest of part 7 (EcoRI+PstI) for future ligations

Gel:

am geldoc 2013-07-04 14hr 41min

Purification of DNA from gel using Qiagen PCR purification kit:

-          like Katrin 25.06.2013

Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

pSB1C3 clone

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

1

6.2

1.35

0.12

2

11.3

1.27

0.21

3

10.2

1.71

0.06

 

 

è DNA frozen away in one of the red boxes with today´s date on the vial.

Clonation of the RBS-GFP-Terminator Construct:

Gel-Extraction of the GFP-Terminator construct from the restriction digest (XbaI+PstI), ligation into opened RBS-Vector (Part 8) and transformation

Gel:

am geldoc 2013-07-04 14hr 39min

Purification of DNA from gel using Qiagen PCR purification kit:

-          like Katrin 25.06.2013

-          Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

GFP-Term clone

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

1

5.4

1.75

0.07

2

19.5

1.62

0.19

3

6.2

1.32

0.10

 

 

è DNA frozen away in one of the red boxes with today´s date on the vial.

Ligation

-Clone 2 was used for ligation to the opened RBS-Vector (also known as part 8 –see 1.7.13)

-ligation was pipetted as follows:

13.5µl HPLC H2O

2µl Ligase buffer

2.5µl RBS-Vector DNA (~20ng)

1µl GFP-Term Clone 2 DNA (~20ng)

1µl Ligase

-For the control, the GFP-Term.-Insert was substituted with HPLC H2O

-Ligation was performed at RT for 10 minutes

-Heat-inactivation was performed at 80°C for 20 minutes

Transformation

-The whole ligation was used for the transformation.

-Transformation performed according to the methods-folder

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Extraction of Terminator and RBS from cultures prepared on 03.07.
Digestion of extracted plasmids containing Terminator and RBS

Extraction of Terminator and RBS from cultures prepared on 03.07.

 

-       extraction of plasmids via NucleoSpinRPlasmid kit

-       concentration measurement on Nanodrop

Sample

Concentration

 

T1

 

69 ng/µl

T2

80 ng/µl

T3

118 ng/µl

T4

95 ng/µl

RBS1

too low

RBS2

52 ng/µl

RBS1

too low

RBS1

too low

 

Digestion of extracted plasmids containing Terminator and RBS

è followed protocol for digestion as written in BioBrickRAssembly Kit

 

                          Terminator:      

Volume

plasmid DNA (pSB1C3)

500ng

PstI

1µl

EcoRI

1µl

NEB cutter buffer (10x)

5µl

H2O

to 50µl

è incubation for 10min at 37°C

è heat inactivation for 20min at 80°C

è store at -20°C

 

M| T1| T2| T3| T4

20130704_terminatorPart7_digest

è Digestion failed

 

è set up digestion again

è add 1µl of PstI and 1µl of EcoRI-HF

è incubation for 45min at 37°C

 

RBS:

Components

Volume

plasmid DNA (pSB1C3)

10µl

PstI

1µl

EcoRI

1µl

NEB cutter buffer (10x)

5µl

H2O

33µl

è incubation for 30min at 37°C

è heat inactivation for 20min at 80°C

è store at -20°C

M |R2 |T1 |T2| T3| T4

20130705_digestion_Terminator_RBS

 

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03th

Restriction Digest of Parts 1-4 for cloning from 1.7.13 - Run on 4% gel

-          0.5 µl uncut plasmid + 3.5 µl dH2O + 1 µl LD 5x

-          Ca. 40 ng of R.D. of plasmids (= 4 µl) supplied with 1 µl of LD 5x

-          Run at 100 V on 4 %-agarose-1xTAE gel; marker: log ladder (3 µl)

-          EtBr staining, destaining, UV detection

Uncut plasmids visible (between 3 and 8kb, although about 2.5kb big), linearized plasmids visible (between 8 and 10 kb…), promoter fragments not visible at all (should be 30-120bps). Will add picture later.

Discussion:

The thick gel might have unrepresentative effects on large fragments (e.g. plasmids). This thesis is supported by the unreadable marker bands between 3 and 8 kb.

The promoter fragments might just be too small to be stained properly or they are not in the constructs. Only sequencing can solve this riddle…

 

Wells used as follows:

M_Part1uncut_Part1RD_Part2uncut_Part2RD_Part3uncut_Part3RD_Part4uncut_part4RD_M

used Marker: NEB Quick-Load 2-Log DNA Ladder (0.1-10kb)

am geldoc 2013-07-03 13hr 40min

Restriction digestion of isolated plasmids from yesterday run on 1% gel (for 1.5h)

can not open scn file. Saved under Reporter team gel doc in folder with this date (gel doc 2013-07-03 15hr 29min, control = 14hr 34 min)

 

 Wells as following:

RiboA (C1,C2,C3), RiboB (C1, C2, C3), RiboC (C1), RiboD (C1, C2, C3), lin part7 (C1, C2, C3), DarR (C1, C2, C4), GFP (C1,C2,C4), pSB1C3 (C1, C2, C3)

same order of wells for control

Control:

am geldoc 2013-07-03 15hr 34min

 

Digested:

am geldoc 2013-07-03 15hr 29min

è GFP-Terminator construct could be cloned successfully (fragment of slightly > 1 kb corresponding likely to GFP (ca. 900 bp) and terminator (ca. 170 bp) visible)

è Clones derived from transformation of linearzised part 7 contain apparently plasmid with part 7 (terminator) à ca. 200 bp fragment visible after digest, corresponding presumably to terminator à clones result possibly from transformation of not linearized plasmids present after incomplete digest of part 7

è For C2 of RiboB cloning, a weak band at ca. 200 bp could be detected, that does not fit with the expected insert of 370 bp à something else got cloned…

è For transformations of ligation reactions containing linearized pSB1C3 as a vector, no transformants containing the desired inserts could be obtained à plasmid that was transformed could be one of the residual template plasmids containing Promoter-RBS-RFP-Terminator (ca. 1.5 kb): after R.D., bands at 1.5 kb and 2 kb corresponding to the expected fragments could be detected. However, band at 1.5 kb is very weak, and for uncut plasmids, a strong band at this height representing the supercoiled plasmid could be detected à it’s also possible that pSB1C3 can’t be restricted properly… anyway, linearized pSB1C3 seems not to work for cloning à cut out terminator from part 7, extract backbone (= linearized pSB1C3) and use it for cloning of Ribo- and DarR biobricks instead…

Sequencing data arrived!

àNo biobrick in the DarR operator construct!

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Transformation of T7 promoter
Cultivation of E.coli containing Terminator and RBS

Transformation of T7 promoter

 

-       use T7 promoter BBa_1712074 in pSB1AK8 backbone (plate 5, 6N)

-       resuspend in 10µl H2O

-       incubation at RT for 5min.

-       transformation of 2µl T7 promoter with 200µl of comp. E.coli cells and incubation for 30min on ice

-       90sec heat shock at 42°C

-       5min on ice

-       add 800µl LB medium and incubate for 1h at 37°C with agitation

-       plate out 100µl of transformation mixture on LB medium containing amikacin

-       centrifuge tubes for 1min, discard 800µl and plate out the rest on new plates

-       incubate plates for 2 days at 37°C

 

Cultivation of E.coli containing Terminator and RBS

-       reporter team cloned terminator (BBa_B0015; pSB1C3 backbone; Cm-resistance) and RBS (BBa_0034; pBS1A2; Amp-resistance) into E.coli

-       inoculation of each 4 liquid cultures in LB containing Cm and Amp

-       incubation at 37°C o/n with agitation

 

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02nd

Plasmid Mini Preparation

Back-up plates

-          for all inoculated clones (except for clones containing plasmids for parts 1 – 4)

-          incubation over day at 37 °C

-          stored at 4 °C

Plasmid Mini Preparation

-          for all inoculated clones

-          kit from Macherey-Nagel

-          elution with pre-warmed 30 µl HPLC water

-          DarR biobrick clone 3: almost all lysate spilled…

-          Concentration measurement on NanoDrop

Concentrations:

concentrations_2

è DarR biobrick clone 3 has strangely high concentration

Test restriction digest of isolated plasmids

è Cut out inserts with EcoRI and PstI (Fermentas/ThermoScientific enzymes, not distribution kit!)

è Only 3 clones tested, if there are 5 to waste less enzyme…

For 1 reaction (10 µl total volume)

0.5 µl PstI

0.5 µl EcoRI

1 µl buffer 0 (both enzymes have 100 % activity and no star activity in this buffer)

3 µl plasmid DNA mix containing ca. 200 – 300 ng plasmid (see picture of NanoDrop table above, rightmost + smallest part of table)

5 µl dH2O

è Master Mix consisting of

12.5 µl PstI

12.5 µl EcoRI

25 µl buffer 0

125 µl dH2O

è In total: 250 µl

è Add 7 µl to each plasmid DNA mix

Incubation for ca. 1 h at 37 °C

Freezing of samples at -20°C

 

Fold ↑

Digestion of plasmid pSB1C3 with EcoRI and PstI from linear precursor
Dephosphorylation of vector pSB1C3
Ligation of Strep-Tag; S. aureus DAC domain purified and S.aureus non purified with pSB1C3
Transformation of ligated samples with competent E.coli cells

Digestion of plasmid pSB1C3 with EcoRI and PstI from linear precursor

 

Components:

Volume

plasmid DNA (pSB1C3)

20µl

PstI

1µl

EcoRI

1µl

NEB cutter buffer (10x)

5µl

H2O

23µl

 

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

 

Dephosphorylation of vector pSB1C3

Components:

Volume

digested pSB1C3

50µl

antarch’s buffer (10x)

5µl

antarch’s phosphatase

1µl

 

Measurement of DNA concentration

Sample

Concentration

pSB1C3 (digested/dephosphorylated)  

5,4ng/µl

Strep-Tag (digested)                             

126ng/µl

S. areus DAC domain (digested)          

181ng/µl

 

Ligation of Strep-Tag; S. aureus DAC domain purified and S.aureus non purified with pSB1C3

Strep-Tag - components:

Volume

insert (Strep-Tag)

1µl

plasmid DNA (pSB1C3)

2µl

T4 ligase

1µl

T4 ligase buffer (10x)

2µl

H2O

14µl

 

S.aureus purified and non purified - components:

Volume

insert (S. aureus DAC domain)

1µl

plasmid DNA (pSB1C3)

2µl

T4 ligase

1µl

T4 ligase buffer (10x)

2µl

H2O

14µl

 

è incubation for 1h at room temperature

Transformation of ligated samples with competent E.coli cells

-       add ligation sample to 200µl of comp. cells and incubate for 30min on ice

è each 2µl and 10µl of ligated Strep-Tag, DAC domain S.aureus and relegation control used

-       90sec heat shock at 42°C

-       5min on ice

-       add 800µl LB medium and incubate for 1h at 37°C with agitation

-       plate out 100µl of transformation mixture on LB medium containing 35µg/ml chloamph.

-       centrifuge tubes for 1min, discard 800µl and plate out the rest on new plates

-       incubate plates for 2 days at 37°C

 

è Transformation failed

 

Fold ↑

01st

Restriction of parts 1 – 4 to cut out promoters for ligation with operator plasmid and restriction of part 8 for inserting GFP-Terminator cassette

Reactions according to manual of Distribution kit:

-          Parts 1 – 3: restriction of 1 µg plasmid DNA with SpeI and EcoRI-HF; 100 µl in total (DNA, enzyme amounts and buffer amount doubled compared to common 50 µl reaction; but distributed to 2 epis with 50 µl each à more complete reaction)

-          Part 4: restriction of ca. 250 ng plasmid DNA (8.7 µl from 29.5 ng/µl solution) with SpeI and EcoRI-HF

-          Part 8: restriction of 500 ng plasmid DNA with SpeI and PstI

-          For parts 1,2, and 3: MasterMix consisting of both enzymes and buffer and sufficient for 4 reactions was used

 

è 20 min at 37 °C digestion

è 20 min at 80 °C heat-kill

 

-          1xTAE-1%agarose gel

-          Load 3 µl of each digest (ca. 30 ng) and 1 µl of plasmid as a control, supplied with 5xDLD

-          3 µl of log ladder as marker

-          Run at 100 V

-          EtBr staining + destaining

Gel:

 

è Still, R.D. is partial à next time: try 30 min incubation

è One can conclude linearization of all plasmids, but promoter fragments are not visible…. Try higher concentrated gel and load higher amounts of DNA

è Reactions stored at – 20 °C

 

Transformation from 27.6.13

-          Colonies grew on all plates except for negative controls and those of pSB1C3 transformation

-          Colonies on without-insert-control of linearized pSB1C3 look slightly pink à remaining template from PCR transformed = pSB1C3 plasmid?

-          Colonies on without-insert-control of linearized part 7 vector might result from not digested plasmid (partial digestion, as usual….)

-          Just 1 clone for Riboswitch biobrick C…

-          Inoculation of 5 clones for each (except for Ribo C) transformation; for without-insert-controls, 3 clones each were inoculated; 4 ml LB+Cm (35 µg/ml)

-          Incubation ON

 

New plasmid preparation for parts 1 – 4

-          Inoculation of parts 1, 3, 4 C1 and part 2 C2 in 4 ml LB+Amp

-          Incubation ON

Re-streak clones for parts 6 and 7 on 35 µg/ml Cm plates + incubation on

Purification of part 8 (RD) products from today

PCR clean-up with Qiagen kit

 

Elution with 50 µl HPLC water (pre-warmed)

NanoDrop:

 

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

part 8 purification

7.5

1.34

1.39

 

 

 

Either just low amounts of DNA in the sample or the purification was not very efficient. Eluded a second time

with 20µl, no DNA found.

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Primer iGEM_17 designed (used for deletion of the restriction site in DAC domain of S. pneumoniae

Fold ↑