Team:Evry/Notebook/Transposons/15.10.14

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14.10.2014 ligation gave only one clone.
 
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<h4> 14.10.2014 ligation </h4>
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<br><br><p>
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it gave only one clone.
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<br><br>
Clone culture:
Clone culture:
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5 ml LB + 5 ul Kan (25 mg/ml) - 37 C°
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<br>5 ml LB + 5 ul Kan (25 mg/ml) - 37 C°
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<br><br></p>
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<br/>It parallel annotation of plasmid pNK2 with Blast is realized.
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<h4> Transformation <i>Pseudovibrio denitrificans</i> & Dh5 alpha with mRFP in BBa_1413044 </h4>
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<br><br><p>
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electrocompetent cells:
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<br><br>
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1 mm cuvette; 1800 V
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<br>recuperation media: MB for <i>Pseudovibrio denitrificans</i> , LB for DH5alpha
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<br><br>
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plating:
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<br><br>
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MB agar for <i>Pseudovibrio denitrificans</i>
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<br>LB agar for LB for DH5alpha
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<br><br></p>
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<h4> Transformation <i>Pseudovibrio denitrificans</i> & Dh5 alpha with BBa_1413001 in BBa_1413044 </h4>
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<br><br><p>
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electrocompetent cells:
 +
<br><br>
 +
1 mm cuvette; 1800 V
 +
<br>recuperation media: MB for <i>Pseudovibrio denitrificans</i> , LB for DH5alpha
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<br><br>
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<br>plating:
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<br><br>
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MB agar for <i>Pseudovibrio denitrificans</i>
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<br>LB agar for LB for DH5alpha
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<br><br></p>
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<div align="center">
<img src="" width="50%"/>
<img src="" width="50%"/>
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</div>
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<br><br>
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<h4> Verification testing of pnK2 in <i>Pseudovibrio denitrificans</i> </h4>
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<br><br><p>
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16  clones of <i>Pseudovibrio denitrificans</i> were grown in 3 ml MB, 30 C°, 24h
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<br><br>
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DNA extraction:
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<br><br>
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Invitrogen DNA extraction kit used with the Gram - protocol.
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<br>nanodrops gave value between 80 ng/ul & 130 ng/ul
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<br><br>
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PCR for Kan gene insertion:
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<br>PCR: enzyme: Q5; template: 200 ng genomic preparation <i>Pseudovibrio denitrificans</i>; oligo: F 82/ R 83; Tm tested: 55; elongation time: 2m00s
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<br><br></p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/6/6c/Kan.JPG" width="50%"/></div>
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<br><br><p>
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PCR for <i>Pseudovibrio denitrificans</i> specific oligo:
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<br>PCR: enzyme: Q5; template: 200 ng genomic preparation <i>Pseudovibrio denitrificans</i>; oligo: F 72/ R 73; Tm tested: 55; elongation time: 2m00s
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<br><br></p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/e/e7/Evry_Pseudo_spe.JPG" width="50%"/></div>
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<br><br><p>
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As a control we tested the specificity of our oligo on the strain we work with:
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<br><div align="center"><img src="https://static.igem.org/mediawiki/2014/0/0b/Pseudo_spe.JPG"width="50%"/></div>
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<br></p>
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<span class="cd-date">Oct 15</span>  
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& <i>Pseudovibrio denitrificans
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</p>
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<span class="cd-date">Aug 08</span>  
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</div>
</div>
</div>
</div>
</html>
</html>

Latest revision as of 23:01, 17 October 2014

Picture

14.10.2014 ligation



it gave only one clone.

Clone culture:
5 ml LB + 5 ul Kan (25 mg/ml) - 37 C°

Transformation Pseudovibrio denitrificans & Dh5 alpha with mRFP in BBa_1413044



electrocompetent cells:

1 mm cuvette; 1800 V
recuperation media: MB for Pseudovibrio denitrificans , LB for DH5alpha

plating:

MB agar for Pseudovibrio denitrificans
LB agar for LB for DH5alpha

Transformation Pseudovibrio denitrificans & Dh5 alpha with BBa_1413001 in BBa_1413044



electrocompetent cells:

1 mm cuvette; 1800 V
recuperation media: MB for Pseudovibrio denitrificans , LB for DH5alpha


plating:

MB agar for Pseudovibrio denitrificans
LB agar for LB for DH5alpha



Verification testing of pnK2 in Pseudovibrio denitrificans



16 clones of Pseudovibrio denitrificans were grown in 3 ml MB, 30 C°, 24h

DNA extraction:

Invitrogen DNA extraction kit used with the Gram - protocol.
nanodrops gave value between 80 ng/ul & 130 ng/ul

PCR for Kan gene insertion:
PCR: enzyme: Q5; template: 200 ng genomic preparation Pseudovibrio denitrificans; oligo: F 82/ R 83; Tm tested: 55; elongation time: 2m00s



PCR for Pseudovibrio denitrificans specific oligo:
PCR: enzyme: Q5; template: 200 ng genomic preparation Pseudovibrio denitrificans; oligo: F 72/ R 73; Tm tested: 55; elongation time: 2m00s



As a control we tested the specificity of our oligo on the strain we work with:


Oct 15

Retrieved from "http://2014.igem.org/Team:Evry/Notebook/Transposons/15.10.14"