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Team:Evry/Notebook/Transposons/15.10.14
From 2014.igem.org
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<h4> 14.10.2014 ligation </h4> | <h4> 14.10.2014 ligation </h4> | ||
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it gave only one clone. | it gave only one clone. | ||
<br><br> | <br><br> | ||
Clone culture: | Clone culture: | ||
<br>5 ml LB + 5 ul Kan (25 mg/ml) - 37 C° | <br>5 ml LB + 5 ul Kan (25 mg/ml) - 37 C° | ||
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<h4> Transformation <i>Pseudovibrio denitrificans</i> & Dh5 alpha with mRFP in BBa_1413044 </h4> | <h4> Transformation <i>Pseudovibrio denitrificans</i> & Dh5 alpha with mRFP in BBa_1413044 </h4> | ||
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electrocompetent cells: | electrocompetent cells: | ||
<br><br> | <br><br> | ||
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MB agar for <i>Pseudovibrio denitrificans</i> | MB agar for <i>Pseudovibrio denitrificans</i> | ||
<br>LB agar for LB for DH5alpha | <br>LB agar for LB for DH5alpha | ||
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<h4> Transformation <i>Pseudovibrio denitrificans</i> & Dh5 alpha with BBa_1413001 in BBa_1413044 </h4> | <h4> Transformation <i>Pseudovibrio denitrificans</i> & Dh5 alpha with BBa_1413001 in BBa_1413044 </h4> | ||
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electrocompetent cells: | electrocompetent cells: | ||
<br><br> | <br><br> | ||
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MB agar for <i>Pseudovibrio denitrificans</i> | MB agar for <i>Pseudovibrio denitrificans</i> | ||
<br>LB agar for LB for DH5alpha | <br>LB agar for LB for DH5alpha | ||
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<img src="" width="50%"/> | <img src="" width="50%"/> | ||
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<br><br> | <br><br> | ||
<h4> Verification testing of pnK2 in <i>Pseudovibrio denitrificans</i> </h4> | <h4> Verification testing of pnK2 in <i>Pseudovibrio denitrificans</i> </h4> | ||
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16 clones of <i>Pseudovibrio denitrificans</i> were grown in 3 ml MB, 30 C°, 24h | 16 clones of <i>Pseudovibrio denitrificans</i> were grown in 3 ml MB, 30 C°, 24h | ||
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PCR for Kan gene insertion: | PCR for Kan gene insertion: | ||
<br>PCR: enzyme: Q5; template: 200 ng genomic preparation <i>Pseudovibrio denitrificans</i>; oligo: F 82/ R 83; Tm tested: 55; elongation time: 2m00s | <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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| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <div align="center"><img src="https://static.igem.org/mediawiki/2014/6/6c/Kan.JPG" width="50%"/></div> |
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PCR for <i>Pseudovibrio denitrificans</i> specific oligo: | PCR for <i>Pseudovibrio denitrificans</i> specific oligo: | ||
<br>PCR: enzyme: Q5; template: 200 ng genomic preparation <i>Pseudovibrio denitrificans</i>; oligo: F 72/ R 73; Tm tested: 55; elongation time: 2m00s | <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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| - | <img src="https://static.igem.org/mediawiki/2014/e/e7/Evry_Pseudo_spe.JPG" width="50%"/> | + | <div align="center"><img src="https://static.igem.org/mediawiki/2014/e/e7/Evry_Pseudo_spe.JPG" width="50%"/></div> |
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| - | </p> | + | As a control we tested the specificity of our oligo on the strain we work with: |
| + | <br><div align="center"><img src="https://static.igem.org/mediawiki/2014/0/0b/Pseudo_spe.JPG"width="50%"/></div> | ||
| + | <br></p> | ||
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<span class="cd-date">Oct 15</span> | <span class="cd-date">Oct 15</span> | ||
</div> | </div> | ||
</div> | </div> | ||
</html> | </html> | ||
Latest revision as of 23:01, 17 October 2014
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
