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WEEK 1
06/01 - 06/05
04-06-15 : Transformation of our biobricks in DH5-alpha competent cells : xxxxxxxx
Materials :
- Competant DH5-alpha cells (100 microL per transformation)
- Ice (in ice bucket/container)
- 2mL tube (1 per a transformation)
- 42°C water bath
- SOC media
- Petri dishes xith LB agar and appropriate antibiotic (2 per transformation)
- 37°C incubator
Protocol :
1/ Thaw on ice one tube of DH5α cells. Place 1.5mL microcentrifuge tubes on wet ice.
2/ Gently mix cells with the pipette tip and aliquot 50µL for each transformation into a 1.5ml centrifuge tube.
3/ Refreeze any unused cells.
4/ Add 1 to 5 µL (1-10 ng) DNA to the cell and mix gently (Not mix by pupetting up and down)
5/ Incubate tubes on ice for 30 min
6/ Heat shock cells for 40s in a 42°C water bath without shaking.
7/ Place tube on ice for 3 minutes.
8/ Add pre-warmed 700µL SOC medium (XXXXXXXX).
9/ Incubate at 37°C for 40 min under shaking condition (200 rpm)
10/ Spread 200 µL of transformed cell on LB + Cm34 (chloramphenicol 34µg/mL).
11/ Incubate dished overnight at 37°C
05-06-15 : Bacterial amplification : xxxxxxxxxxx
Materials :
• Petri dishes
• LB with or without chlroramphénicol
• 15mL falcon tubes (3 per Petridish used)
Protocol :
1/ Fill the tubes with 5mL of the correct media (3 tubes per strain)
2/ Pick three transformed clones per Petri box (1 clone per 15mL tube)
3/ Incubate in agitation (8 hours at 200 rpm, 37°C)
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