Difference between revisions of "Team:Munich/engineering2.html"

 
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<html>
 
<div class="event-info">
 
<div class="event-info">
  
−
<h4>Transforming E. Coli Rosetta and MG1655 for pRED/ET engineering</h4>
+
<h4>Transforming E. Coli Rosetta and MG1655 for pRED/ET Engineering</h4>
 
<em>2018/08/28</em>
 
<em>2018/08/28</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
Line 7: Line 8:
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Dominic</td>
+
       <td>Dominic Schwarz</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>epo trafo</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/c/ca/T--Munich--WL1_Electrocompetent_transformation.pdf" target="_blank">Electrocompetent transformation</a>
 +
</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>MG1655 is E.Coli WT as backup
+
       <td>Genetic engineering is planned in E. Coli Rosetta, MG1655 is E.Coli WT as backup.
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td>colonies only on MG1655 plate
+
       <td>Colonies only on E. Coli MG1655 plate;
−
No colonies on E.coli Rosetta
+
No colonies on E. Coli Rosetta
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
 +
<h4>pRED/ET Genome Engineering of Delta msb-B and Delta recBCD Strains</h4>
 +
<em>2018/08/29</em>
 +
<table class="table table-borderless">
 +
    <tr>
 +
 +
      <td>Participants:</td>
 +
      <td>Dominic Schwarz</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Protocol:</td>
 +
      <td>
 +
<a href="https://static.igem.org/mediawiki/2018/b/bf/T--Munich--pRED_ET-Ecoli_Gene_Deletion_Kit.pdf.pdf" target="_blank">pRED/ET engineering</a>
 +
 +
</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Notes:</td>
 +
      <td>The template for the resistance cassette for deletions was taken from a plasmid containing mRFP
 +
</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 +
      <td>Red colonies. Elena (Advisor) and Dominic decided to repeat the experiment but to do a dpnI digest before to get rid of the template DNA.
 +
</td>
 +
    </tr>
 +
</table>
  
−
<h3>8/29</h3>
+
<h4>Transforming E. Coli DH5a to Find a Reason for the Contamination</h4>
−
<h4>pRED/ET genome engineering of delta msb-B and delta recBCD strains</h4>
+
<em>2018/08/30</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Dominic</td>
+
       <td>Dominic Schwarz</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>pRED/ET engineering protocol</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/c/ca/T--Munich--WL1_Electrocompetent_transformation.pdf" target="_blank">Electrocompetent transformation</a>
 +
</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>the template for the resistance cassette for deletions was taken from a plasmid containing mRFP
+
       <td>CAP_recBCD, CAP_msbb, psb1c3_mrfp in Dh5a
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td>red colonies
+
       <td>Red colonies on all plates -> mRFP contamination
−
Redoing the experiment but doing a dpni digest before
+
</td>
 +
    </tr>
 +
</table>
  
 +
<h4>Creating a Selection Cassette from pSB1C3</h4>
 +
<em>2018/08/30</em>
 +
<table class="table table-borderless">
 +
    <tr>
 +
 +
      <td>Participants:</td>
 +
      <td>Dominic Schwarz</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Protocol:</td>
 +
      <td>
 +
<a href="https://static.igem.org/mediawiki/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restrition digest</a>,
 +
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>
 +
</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Notes:</td>
 +
      <td>DpnI
 +
</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 +
      <td>CAP_recBCD 18ng/ul
 +
CAP_msbB 12ng/ul
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
−
<h3>8/30</h3>
+
<h4>pRED/ET Genome Engineering of Delta msb-B and Delta recBCD Strains</h4>
−
<h4>Transforming E. Coli DH5a to find a reason for the contamination</h4>
+
<em>2018/09/01</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Dominic</td>
+
       <td>Dominic Schwarz</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>epo trafo</td>
+
       <td><a href="https://static.igem.org/mediawiki/2018/b/bf/T--Munich--pRED_ET-Ecoli_Gene_Deletion_Kit.pdf.pdf" target="_blank">pRED/ET engineering</a></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>CAP_recBCD, CAP_msbb, psb1c3_mrfp in Dh5a
+
       <td>The template for the resistance cassette for deletions was taken from a plasmid containing mRFP
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
−
       <td>Results:</td>
+
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td>red colonies on all plates -> mRFP contamination
+
       <td>Colonies on both plates  
 +
 
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
 +
 +
<h4>Verifying Deletion Strains of E. Coli MG1655</h4>
 +
<em>2018/09/05</em>
 +
<table class="table table-borderless">
 +
    <tr>
 +
 +
      <td>Participants:</td>
 +
      <td>Dominic Schwarz</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Protocol:</td>
 +
      <td>
 +
<a href="https://static.igem.org/mediawiki/2018/8/88/T--Munich--WL1_Colony_PCR.pdf" target="_blank">Colony PCR</a>,
 +
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>
 +
</td>
 +
    </tr>
 +
    <tr>
 +
      <td>Notes:</td>
 +
      <td>Primers: VF2, geno_msb-B_rv, geno_recBCD_rv;
 +
Ta: 48°C
 +
t= 1kb/min <br>
 +
RecBCD: 1,2,3,4,5 <br>
 +
msbB: 35,36,37,38,39
 +
expected: RecBCD 443bp
 +
Expected: msb-B 574bp
 +
</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 +
      <td>All 5 picked colonies were positive for the insertion of the selection cassette PIC
 +
</td>
 +
    </tr>
 +
</table>
 +
 +
<figure class="figure">
 +
  <img src="https://static.igem.org/mediawiki/2018/b/b3/T--Munich--Labbook_20180905_geno_analgel_recBCD12345%28443kb%29_msbB678910%28574kb%29.jpeg" class="figure-img img-fluid rounded" alt="awesome gel.">
 +
    <figcaption class="figure-caption">sick caption</figcaption>
 +
    </figure>
  
 
</div>
 
</div>
 +
</html>

Latest revision as of 00:25, 18 October 2018

Transforming E. Coli Rosetta and MG1655 for pRED/ET Engineering

2018/08/28
Participants: Dominic Schwarz
Protocol: Electrocompetent transformation
Notes: Genetic engineering is planned in E. Coli Rosetta, MG1655 is E.Coli WT as backup.
Results: Colonies only on E. Coli MG1655 plate; No colonies on E. Coli Rosetta

pRED/ET Genome Engineering of Delta msb-B and Delta recBCD Strains

2018/08/29
Participants: Dominic Schwarz
Protocol: pRED/ET engineering
Notes: The template for the resistance cassette for deletions was taken from a plasmid containing mRFP
Results: Red colonies. Elena (Advisor) and Dominic decided to repeat the experiment but to do a dpnI digest before to get rid of the template DNA.

Transforming E. Coli DH5a to Find a Reason for the Contamination

2018/08/30
Participants: Dominic Schwarz
Protocol: Electrocompetent transformation
Notes: CAP_recBCD, CAP_msbb, psb1c3_mrfp in Dh5a
Results: Red colonies on all plates -> mRFP contamination

Creating a Selection Cassette from pSB1C3

2018/08/30
Participants: Dominic Schwarz
Protocol: Restrition digest, PCR purification
Notes: DpnI
Results: CAP_recBCD 18ng/ul CAP_msbB 12ng/ul

pRED/ET Genome Engineering of Delta msb-B and Delta recBCD Strains

2018/09/01
Participants: Dominic Schwarz
Protocol: pRED/ET engineering
Notes: The template for the resistance cassette for deletions was taken from a plasmid containing mRFP
Results: Colonies on both plates

Verifying Deletion Strains of E. Coli MG1655

2018/09/05
Participants: Dominic Schwarz
Protocol: Colony PCR, Agarose gel
Notes: Primers: VF2, geno_msb-B_rv, geno_recBCD_rv; Ta: 48°C t= 1kb/min
RecBCD: 1,2,3,4,5
msbB: 35,36,37,38,39 expected: RecBCD 443bp Expected: msb-B 574bp
Results: All 5 picked colonies were positive for the insertion of the selection cassette PIC
awesome gel.
sick caption