Team:Evry/Notebook/Biology/08-15-2014

Title

BBa_E0240 and BBa_I20260 are respectively into a PSB1C3 and a PSB1K3 vector. In order to select colonies, LB complemented with kanamycin or chloramphenicol are necessary. 200 ml of LB agar medium was prepared with 7 g of LB Agar powder(Sigma) into 200 ml of miliQ water. After complete dissolution, the solution was sterilized in the Tuttmauer 2540ML apparatus, STE 20 minutes at 121°C and EXT+DRY 15 minutes. 50 µl of kanamycin stock solution (25 mg/L) was added to 50 ml of LB agar to pour 2 plates. 150 µl of chloramphenicol stock solution was added in the 150 LB agar remaining ml to pour 6 plates.
The transformation was performed on DH5 alpha ''E. coli'', as followed:

1. Remove E. coli competent tubes from -80°C and keep it on ice
2. Add 1 µl of template (here solubilized plasmids from the registry distribution kit) and mix gently
3. Incubate 10 minutes on ice
4. Perform an heat shock 30 seconds at 42°C
5. Incubate 2 minutes on ice
6. Add 3 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
7. Plate 200 µl of BBa_E0240 on a chloramphenicol LB agar plate and BBa_I20260 on a Knamycin LB agar plate
8. Incubate plate overnight at 37°C