IGEM:Paris Bettencourt 2012/Notebooks/Delay group/day by day//2012/07/26

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Incubate 1h at 22°C (PCR machine)
Incubate 1h at 22°C (PCR machine)
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==transformation==
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The product of the ligation is transformed on Amp and Kan plates (3µl of the ligation mix for each). PSC001(Amp) is used as positive control (1µl). I forgot the negative control.
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Recovery

1- pLac-RBS-GFP-TT clone 1 (From Antoine) +IPTG+Amp

2- pLac-RBS-GFP-TT clone 1 (From Antoine) +Amp

3- pLac-RBS-GFP-TT clone 1 (From Antoine) +IPTG+Kan

4- pLac-RBS-GFP-TT clone 1 (From Antoine) +Kan

5- pLac-RBS-GFP-TT clone 1 (From Denis) + IPTG + Amp

6- pLac-RBS-GFP-TT clone 1 (From Denis) + Amp

7- araC-pBad-RBS-LacI clone 2 (from the 2009 Paris iGEM team) + Amp

8- araC-pBad-RBS-LacI clone 2 (from the 2009 Paris iGEM team) + Kan

9- pLac-RBS-GFP-TT clone 2 (From Antoine) +IPTG+Amp

10- pLac-RBS-GFP-TT clone 2 (From Antoine) +Amp

11- pLac-RBS-GFP-TT clone 4 (From Antoine) +IPTG+Amp

12- pLac-RBS-GFP-TT clone 4 (From Antoine) +Amp

We'll need to do a glycerol stock for the strains that worked.

Digestion

insert = pLac-GFP-TT

vector = araC-pBad-RBS-LacI

The insert is digested in E&S, the vector is digested in E&X.

Digestion of the insert :

  • 13µl insert (150 ng/µl)
  • 2µl EcoRI
  • 2µl SpeI
  • 2µl FD Green Buffer
  • 1µl H2O

Digestion of the vector :

  • 11µl vector (180 ng/µl)
  • 2µl EcoRI
  • 2µl XbaI
  • 2µl FD Buffer
  • 3µl H2O

The insert is purified on a 0.8% agarose gel. We obtain a concentration of 24.1 ng/µl

The vector is purified wth the PCR kit. We obtain a concentration of 86.4 ng/µl

ligation

insert/vector~5

In 10µl :

  • 3µl of insert (86.4 ng/µl)
  • 0.6µl of vector (24.1 ng/µl)
  • 0.5µl ligase
  • 1µl buffer
  • 4.9µl H20

Incubate 1h at 22°C (PCR machine)

transformation

The product of the ligation is transformed on Amp and Kan plates (3µl of the ligation mix for each). PSC001(Amp) is used as positive control (1µl). I forgot the negative control.

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