Team:Paris Liliane Bettencourt/Notebook/2011/08/23/

From 2011.igem.org

Team IGEM Paris 2011

Cyrille

going on with TetArray

We are going to ligate a promoter pVeg-SpoVG in front of the TetR-YFP protein, even if this is not biobricked. (several PstI sites will have to be eliminated)

The promoter will have to be inserted into the holding plasmid. Since the fragment is small, we will amplify it by PCR.

Dilution of the primers:

  • 2 µL of primer VF2 at 100µM in 6µM of H2O (25µM final)
  • 2 µL of primer VR at 100µM in 6µM of H2O (25µM final)


PCR:

Tube one or 2

  • 25 µL of PCR Mastermix
  • 2µL of S24 DNA (the promoter)
  • 2µL of primer VF2 (1µM final)
  • 2µL of primer VR (1µM final)
  • 19µL of H2O

Control tube

  • 25 µL of PCR Mastermix
  • 2µL of S24 DNA (the promoter)
  • 23µL of H2O


Temperature

  • 95°C 2 min


  • 95°C 30 sec
  • 55°C 30 sec
  • 50°C 30 seq
  • 72°C 1 min

Repeat 40 times

  • Final extension 72°C 5min

The gel was runned and bands cutted

ladder- ctrl - - bands



Camille

A gel to verify the PCR products show that the PCR worked. Therefore I launched cultures of the different strains I wanted to clon the promoter in.



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