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

 
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<html>
 
<div class="event-info">
 
<div class="event-info">
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<h4>Assembling Biobrick for RecB, C, D</h4>
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<em>2018/08/31 – 2018/09/07</em>
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<table class="table table-borderless">
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    <tr>
 +
 +
      <td>Participants:</td>
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      <td>Enikö Baligács</td>
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    </tr>
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    <tr>
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      <td>Protocol:</td>
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      <td>
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<a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>,
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<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>,
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<a href="https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020" target="_blank">Gel extraction</a>,
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<a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,
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<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a>,
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<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Mini Prep</a>
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</td>
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    </tr>
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    <tr>
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      <td>Notes:</td>
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      <td> Three PCRs for three BioBricks with primers: RecD_BB_EcoRI_fw & RecD_BB_SpeI_rv (expected: 3,5 kb)
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RecB_BB_EcoRI_fw & RecB_BB_SpeI_rv (expected: 1,8 kb)
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RecC_BB_EcoRI_fw & RecC_BB_SpeI_rv (expected: 3,3 kb)
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AT 68° ET 70 s; restriction digest of pSB1C3_mRFP with EcoRI and SpeI and PCR purification of the Backbone each PCR was digested with EcoRi and SpeI and ligated into the Backbone.
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</td>
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    </tr>
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<tr>
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      <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
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      <td> correct bands were cut out. Ligations gave colonies. Test PCR and sequencing confirmed ligation, sent more primers for complete insert sequencing. Completely sequenced positive clones were obtained.
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<figure class="figure">
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  <img src="https://static.igem.org/mediawiki/2018/c/c1/T--munich--2018-08-31_at_19.07.13.jpeg " class="figure-img img-fluid rounded" alt=" ">
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    <figcaption class="figure-caption"></figcaption>
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    </figure>
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</td>
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    </tr>
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</table>
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<h3>8/31</h3>
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<h4>Removing PstI Restriction Site from pSB1C3_RecB</h4>
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<h4>Assembling biobrick for RecB, C, D</h4>
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<em>2018/09/17-2018/10/02</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
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       <td>eni, katja</td>
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       <td>Enikö Baligács</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
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       <td>PCR, agarose gel, gel extraction, ligation, chem trafo, miniprep </td>
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       <td>
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<a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>,  
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<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,  
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<a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,  
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<a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson assembly</a>,  
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<a href="https://static.igem.org/mediawiki/2018/f/fc/T--Munich--Transformation01.pdf" target="_blank">Transformation</a>
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</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>RecD_BB_EcoRI_fw & RecD_BB_SpeI_rv (3,5 kb) <br>
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       <td>Because of time restraints, this experiment was done using ligation and gibson assembly approaches in parallel <br>
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RecB_BB_EcoRI_fw & RecB_BB_EcoRi_rv (1,8 kb) <br>
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PCR: RecB_BB_PstI-remove-Lig
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RecC_BB_EcoRI_fw & RecB_BB_EcoRI_rv (3,3 kb) <br>
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Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv
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AT 68° ET 70 s
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Templ: RecB_BioBrick Plasmid
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AT: 72° ET: 11 min
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<br>
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PCR: RecBCD_BB_PstI_remove_lig
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Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv
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Templ: RecBCD_BioBrick Plasmid
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AT: 72° ET: 11 min
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<br>
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PCR: RecBCD-TEV_BB_PstI_remove-Lig
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Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv
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Templ: RecBCD-TEV_BioBrick Plasmid
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AT: 72° ET: 11 min
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<br>
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PCR: RecB_BB_PstI-remove-GA
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Primer: RecB_mut_fw & RecB_mut_rv
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Templ: RecB_BioBrick Plasmid
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AT: 72° ET: 11 min
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<br>
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PCR: RecBCD_BB_PstI_remove_GA
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Primer: RecB_mut_fw & RecB_mut_rv
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Templ: RecBCD_BioBrick Plasmid
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AT: 72° ET: 11 min
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<br>
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PCR: RecBCD-TEV_BB_PstI_remove-GA
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Primer: RecB_mut_fw & RecB_mut_rv
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Templ: RecBCD-TEV_BioBrick Plasmid
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AT: 72° ET: 11 min
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
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       <td>Results:</td>
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       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
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       <td>PIC only one worked, but somehow all were ligated :D</td>
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       <td>Colonies were obtained, but sequencing showed incorrect cut site assembly. Because of time restrictions these attempts were not continued and we focused on other parts of the project.
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</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
 
</div>
 
</div>
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</html>

Latest revision as of 00:39, 18 October 2018

Assembling Biobrick for RecB, C, D

2018/08/31 – 2018/09/07
Participants: Enikö Baligács
Protocol: PCR, Agarose gel, Gel extraction, Ligation, Chemical transformation, Mini Prep
Notes: Three PCRs for three BioBricks with primers: RecD_BB_EcoRI_fw & RecD_BB_SpeI_rv (expected: 3,5 kb) RecB_BB_EcoRI_fw & RecB_BB_SpeI_rv (expected: 1,8 kb) RecC_BB_EcoRI_fw & RecC_BB_SpeI_rv (expected: 3,3 kb) AT 68° ET 70 s; restriction digest of pSB1C3_mRFP with EcoRI and SpeI and PCR purification of the Backbone each PCR was digested with EcoRi and SpeI and ligated into the Backbone.
Results: correct bands were cut out. Ligations gave colonies. Test PCR and sequencing confirmed ligation, sent more primers for complete insert sequencing. Completely sequenced positive clones were obtained.

Removing PstI Restriction Site from pSB1C3_RecB

2018/09/17-2018/10/02
Participants: Enikö Baligács
Protocol: PCR, PCR purification, Ligation, Gibson assembly, Transformation
Notes: Because of time restraints, this experiment was done using ligation and gibson assembly approaches in parallel
PCR: RecB_BB_PstI-remove-Lig Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv Templ: RecB_BioBrick Plasmid AT: 72° ET: 11 min
PCR: RecBCD_BB_PstI_remove_lig Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv Templ: RecBCD_BioBrick Plasmid AT: 72° ET: 11 min
PCR: RecBCD-TEV_BB_PstI_remove-Lig Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv Templ: RecBCD-TEV_BioBrick Plasmid AT: 72° ET: 11 min
PCR: RecB_BB_PstI-remove-GA Primer: RecB_mut_fw & RecB_mut_rv Templ: RecB_BioBrick Plasmid AT: 72° ET: 11 min
PCR: RecBCD_BB_PstI_remove_GA Primer: RecB_mut_fw & RecB_mut_rv Templ: RecBCD_BioBrick Plasmid AT: 72° ET: 11 min
PCR: RecBCD-TEV_BB_PstI_remove-GA Primer: RecB_mut_fw & RecB_mut_rv Templ: RecBCD-TEV_BioBrick Plasmid AT: 72° ET: 11 min
Results: Colonies were obtained, but sequencing showed incorrect cut site assembly. Because of time restrictions these attempts were not continued and we focused on other parts of the project.