Paris/July 16

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== Transformations of Biobricks ==

Revision as of 07:59, 17 July 2007


Contents

Plasmid pKs::DGAT expression in E. Coli

We tried to see TG with NR died on E. Coli cells transfected with pKs::DGAT, an IPTG-inducible promoter. We tried different growth media containing more or less oleate, that should in theory increase TG synthesis.
Paris EColi DGAT.jpg
Results : we don't see the inductible effect of IPTG. We can think that :

  • Either the fluorescence without IPTG is due to a leak of the promoter
  • Either DGAT is not induce in presence of IPTG, and the fluorescence we see is only a background.

Growth kinetics of w121 strain

Results of the previous day : we lost everything because of a crash of the computer :( Sorry Eimad !

Transduction of MG1655 with P1 stock made on w121

  • Control (1mL LB MgSO4 30mM; CaCl2 15mM)
  • 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
  • 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
  • 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON

=>For the nth time, it is not working : we have only contaminants.


MiniPreps

  • I0500 clones 1, 2
  • pJ23107 clones 1, 2

PCR purification

  • Lox71-FtsZ1
  • FtsZ2
  • DGAT1
  • DGAT2

PCRs

The Lox66-DapAColi PCR did not work... We'll try again with different annealing temperature

Assembly PCRs:

  • Lox71-FtsA-FtsZ-1 + FtsZ-2
  • DGAT-1 + DGAT-2


The Lox66-DapAColi PCR did not work... so we try a gradient of annealing temperatures

PCR : Lox66-DapAColi
Name Lox66-DapAColi
Annealing T° 50-65°C
Time of elongation 2m00'
Number of Cycles 35
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 6 Lox66-DapAColi-F 10µM 2.5µL
oligoR 7 DapAColi-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toothpick in glycerol stock of MG1655


Transformations of Biobricks