ULB/13 August 2009

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== August 13, 2009 ==
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==Manipulation on plasmides==
==Manipulation on plasmides==

Revision as of 09:56, 1 October 2009

iGEM Team:ULB-Brussels Wiki


Manipulation on plasmides

  • Bacteria with the plasmid with RBS (BBa_B0034) growed.
  • The ligation of the promoter-RBS-plasmid (chloramphenicol resistant) didn't work: the reason could be the use of the protocol of manual assembly, who told us to leave league 10 minutes at room temperature, then deactivate the enzymes, insted of 24 hours by the T4 ligase guide.
  • Vérification que les bricks sont présent dans le milieu dans nos extractions : Digestion du promoteur, promoteur+RBS, RBS+RFP et le terminateur par les enzymes de restrictions EcoRI et Spel pour les 2 premiers et Xbal et Pstl pour les autres.
  • Verify that the bricks are now in the medium of our extractions: Digestion of the promoter and promoter + RBS by restriction enzymes EcoRI and Spel, RBS + RFP and the terminator by XbaI and PstI.
  • We made a PCR with primers for ccdA program received at 55 ° C then a gel migration.

Results

les puits du dessus  : Promoteur1, promoteur2, RBS+RFP1, RBS+RFP2, promoteur+RBS1, promoteur+RBS2, terminateur1, terminateur2.les puits du dessus : les résultats des migations des PCR

  • Verification of bricks:4 DNA primers "normal", white primers "normal", 4 DNA primers reverse, white reverse primers.
  • PCR: all tubes have succeeded except the one control sample which is out of place (4th position instead of 6th).

</div>

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