Team:Marburg/Notebook:August

From 2013.igem.org

(Difference between revisions)
 
(7 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:Marburg/Template:Header}}
{{:Team:Marburg/Template:Header}}
-
{{:Team:Marburg/Template:ContentTitle}}
+
{{:Team:Marburg/Template:ContentTitleNav}}
-
Notebook: August
+
Notebook: August <html><a href="https://2013.igem.org/Team:Marburg/Notebook:September"><img src="https://static.igem.org/mediawiki/2013/7/71/Mr-igem-next-arrow.png" style="float:right;margin-left:5px !important;" alt="Next"></a>&nbsp;<a href="https://2013.igem.org/Team:Marburg/Notebook:July"><img src="https://static.igem.org/mediawiki/2013/1/13/Mr-igem-previous-arrow.png" alt="Previous" style="float:right;"></a></html>
-
{{:Team:Marburg/Template:ContentStart}}
+
{{:Team:Marburg/Template:ContentStartNav}}<html>
-
 
+
-
<html>
+
-
<!-- HIER -->
+
<div class="notebooky-entry">
<div class="notebooky-entry">
Line 40: Line 37:
<p><ul class="digest">
<p><ul class="digest">
<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
-
<li>0.3 µl EcoRI</li>
+
<li>0.3 µl <i>Eco</i>RI</li>
-
<li>0.3 µl PstI</li>
+
<li>0.3 µl <i>Pst</I></li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
-
<table class="gel digest">
+
<p>After that, a gel electrophoresis was made, but failed and has to be repeated the next day</p>
-
<colgroup>
+
-
<col width="50%" />
+
-
<col width="50%" />
+
-
</colgroup>
+
-
<thead>
+
-
<tr>
+
-
<th colspan="2" class="title">Gel electrophoresis</th>
+
-
</tr>
+
-
</thead>
+
-
<tbody>
+
-
<tr>
+
-
<td>
+
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
-
</td>
+
-
<td>
+
-
<p>
+
-
<span class="gel-elc">Gel substances</span>
+
-
<ul class="gel-sub">
+
-
<li>1% Agarose gel</li>
+
-
<li>10 µl RedSafe in 50 ml gel</li>
+
-
<li>x µl Hyper Ladder</li>
+
-
</ul>
+
-
</p>
+
-
<p>
+
-
<span class="exp">Expactations</span>
+
-
<ul class="exp">
+
-
<li>Lane 1: 5300 kbp</li>
+
-
<li>Lane 2: 3300 kbp</li>
+
-
<li>Lane 3: 1300 kbp</li>
+
-
</ul>
+
-
</p>
+
-
<p><font color="#FF0000">After that, a gel electrophoresis was made, but failed and has to be repeated the next day</font></p>
+
-
</td>
+
-
</tr>
+
-
</tbody>
+
-
</table>
+
</div>
</div>
</fieldset>
</fieldset>
Line 134: Line 95:
<div class="exp-content">
<div class="exp-content">
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.<br />
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.<br />
-
The plates were incubated over night at 37° C.</p>
+
The plates were incubated over night at 37 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 172: Line 133:
<p><ul class="digest">
<p><ul class="digest">
<li>1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)</li>
<li>1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)</li>
-
<li>0.3 µl EcoRI</li>
+
<li>0.3 µl <i>EcoR</i>I</li>
-
<li>0.3 µl PstI</li>
+
<li>0.3 µl <i>PstI</i></li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 191: Line 152:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/2/26/Gel_2013-08-02.png" width="60%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 199: Line 160:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
</ul>
</ul>
-
</p>
 
-
<p>
 
-
<span class="exp">Expactations</span>
 
-
<ul class="exp">
 
-
<li>Lane 1: 5300 kbp</li>
 
-
<li>Lane 2: 3300 kbp</li>
 
-
<li>Lane 3: 1300 kbp</li>
 
-
</ul>
 
-
</p>
 
<p>Worked as expected.</p>
<p>Worked as expected.</p>
</td>
</td>
Line 240: Line 192:
<li>pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.</li>
<li>pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.</li>
</ul>
</ul>
-
<p>All plasmids are brought to Marian who will transform them in <i>P. tricornutum</i> cells.</p>
+
<p>All plasmids are brought to Marian who will transform them into <i>P. tricornutum</i> cells.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 247: Line 199:
</html>
</html>
-
{{:Team:Marburg/Template:ContentEnd}}
+
{{:Team:Marburg/Template:ContentEndNav}}
{{:Team:Marburg/Template:Footer}}
{{:Team:Marburg/Template:Footer}}

Latest revision as of 11:15, 27 October 2013

Notebook: August Next Previous

01.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

2 additional sequence samples were sent out for sequencing.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
  • 0.3 µl EcoRI
  • 0.3 µl Pst
  • 1 µl CutSmart
  • 7.4 µl ddbidest. H2O

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

After that, a gel electrophoresis was made, but failed and has to be repeated the next day

Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

02.08.2013

Sequencing
Investigator: Dominik
Aim: Analysis of the sequence of pSB1A3-iBB496315.

Obviously the wrong plasmid was sent out for sequencing. Due to the lack of enough plasmids for an additional sequence analysis, we will redo a new miniprep.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.
The plates were incubated over night at 37 °C.

03.08.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmid pSB1A3-iBB496315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB496315 and iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddbidest. H2O

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

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)

Worked as expected.

14.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of sequence analysis of the plasmids pSB1A3-iBB496315, pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

  • pSB1A3-iBB496315: Okay
  • pSB1A3-iBB4+iBB10+iBB96315: mutation in signal peptide that leads to Trp → Leu. Will be ignored
  • pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.

All plasmids are brought to Marian who will transform them into P. tricornutum cells.

Retrieved from "http://2013.igem.org/Team:Marburg/Notebook:August"