Team:Paris Liliane Bettencourt/Notebook/2011/08/01/

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Team IGEM Paris 2011

Contents

Cyrille

Quick change mutagenesis of pHM3

Facing the problems with the mutagenesis the last week, I decided to come down to the original protocole, and try another time with all the keys in hand. The original protocol from the stratagene kit was downloaded from the web. Following its advises, I came down with the new protocol.

The used primers have for sequence: GAT CCT TTT TTT ATA ACA GGC TTT CAC TGG CCG TCG TTT TAC AAC G and the reverse complement, that change the codon in blod in the original sequence. GAT CCT TTT TTT ATA ACA GGA ATT CAC TGG CCG TCG TTT TAC AAC G .

The theoretical melting point and annealing point of the whole primer, calculated in the NEB in the GC buffer is:

  • Annealing temperature: 72°C
  • Tm: 81°C

The Stratagene guideline indicates that we have to remove 1°C per %mismatch. We have 2 mismatch, which rise down the melting temperature of 4 degree. We will add some DMSO in the PCR. 1% of DMSO raise down the Annealing temperature or 0.6°C. We will go to 3% DMSO to help the PCR.

The new temperatures are:

  • Annea. temp: 70.2°C
  • Tm: 77°C

The new protocol, using the Stratagene reference protocol will be:

Prepare a 1/100 dilution of the 991 ng/μL (9.91 ng/μL)

  • 10x GC polymerase buffer: 5μL
  • DMSO 100%  : 1.5μL (3% final)
  • dNTP (10mM)  : 1 μL
  • pHM3 (9.91 ng/μL)  : 2-5-10 μL (20 ng final)
  • Primer fwd ( 1μM = 15,18 ng/μL) : 8,2 μL (125ng)
  • Primer rev ( 1μM = 15,18 ng/μL) : 8,2 μL (125 ng)
  • H2O  : 23,1-20,1-13,1 μL
  • Phusion (2U/μL)  : 1 μL

This time, we will do 3 attempts, and the control will be the same but without the dNTP.

So I'll prepare a mastermix that will be aliquoted in 4 different PCR tubes:

  • 10x GC polymerase buffer: 20μL
  • DMSO 100%  : 6μL
  • Primer fwd  : 32,8 μL
  • Primer rev  : 32,8 μL

Aliquot in the 4 tubes, and complete with the missing DNA, water and phusion.

Then the cicles will be: 

1- 95°C 5 min
2- 95°C 1 min
3- 70°C 1 min
4- 55°C 1min
5- 72°C 12 min
6- goto 2 15 times
7- 72°C 20 min (to complete unfinished polymerisation or ligation)
8- 4°C Forever

Evrything was placed in the PRC machine at 13:00 today. The PCR machine crashed 3 times because of the heat. So at five, the PCR was done again, with another machine. The transformation will be done tomorrow.

Axel

Transformation of E. coli

ligation of pHM3 plasmid with k143079 (it works): we have to select one colony and make a glycerol of it and transform it into subtilis (useful for the modelers)

Kevin

Culture of double transformated cells

Diluted at 1/100 => expected to be between OD 300 and 600

  • 3 Tubes pSG20/pDAG464 at 37°C
  • 3 Tubes pFX234/pDAG479 at 37°C

At OD 300-600, induction of fusion protein with arabinose 0%, 0,1 % and 0,2% for 1h30.

Microscopy

LacI/LacO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.
tetR/tetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.



Camille

T7 polymerase amber transformation succeeded, we then launched it in liquid culture to prepare glycerols.







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