Team:Evry/Notebook/Transposons/16.10.14

From 2014.igem.org

(Difference between revisions)
Line 18: Line 18:
PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s
PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s
<br><br></p>
<br><br></p>
-
<div align="center">
+
 
-
<img src="https://static.igem.org/mediawiki/2014/b/b9/Evry_Is_ecoli.JPG" width="50%"/></div>
+
<img src="https://static.igem.org/mediawiki/2014/b/b9/Evry_Is_ecoli.JPG" width="50%"/>
<br><br>
<br><br>
<h4> defining where the transposase integrate </h4>
<h4> defining where the transposase integrate </h4>
Line 39: Line 39:
</p>
</p>
<br><br>
<br><br>
-
<div align="center">
+
 
-
<img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG" width="50%"/></div>
+
<img src="https://static.igem.org/mediawiki/2014/a/af/Evry_Genome_cut_religated.JPG" width="50%"/>

Revision as of 22:54, 17 October 2014

Picture

Show BBa_1413044 work even empty in DH5alpha



8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control

Preparation for PCR colony:
Spin down 500 ul culture at 14000 g - 2 min, resuspend in 100 ul H20 MilliQ, put at 95 C° for 10 min then spin down at 14000 g - 2 min.

PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s



defining where the transposase integrate



find enzyme with a good cut numbers in the genome and not in our insert: HindIII Digestion of 8 Pseudovibrio denitrificans DNA preparation by HindIII
50ul final volume, 200 ng DNA preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul HindIII. 1h

Then religate:
Ligation: 20 ul final volume, 2 ul 10x T4 ligase buffer, 1 ul T4

then light digestion with XbaI
50ul final volume, 20 ul DNA religated preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul XbaI enzyme. 15 min
deactivation at 80 C° for 20 min

then PCR
PCR: enzyme: Q5; template: 3 ul of digestion by XbaI; oligo: F 105/ R 106; Tm tested: 55; elongation time: 4m00s



Oct 16