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Team:Paris Liliane Bettencourt/Notebook/2011/07/29/
From 2011.igem.org
Contents |
Cyrille
QCM
The transformation of the quick change has given a few clones on the plates where 200 muL was spread. This show again the poor quality of our competent cells. 10 of the most promising clones ( 8 from the tube 1 ) where taken and put in culture in 15 ml of LB+Amp. The miniprep and the digestion experiments is planned for the evening.
MH1 competent cells preparation
20 MuL of MH1 competent cells taken from the Hervé le Hir laboratory where put in culture in 15 mL of LB medium for 3 hours. Two of the tubes will serve to make glycerols of the cells, whereas the two other serves as preculture for the competent production.
The protocol for the competent cells is the following:
- Take 1mL of precultured cell in 200 mL. Let them growth for 2-3h until they reach the OD600 between 0,3 and 0,6.
- Spin the culture 10 min at 4000 rpm
- Resuspend the cells in 100mL of CaCl2 at 50 mM ( 5 mL CaCL2 1M qsp 100 mL, and filtered )
- Leave 20 min in ice
- Spin 10 min at 4000rpm at 4°C
- Resuspend in 20mL of CaCl2-Glycerol-Eau ( 1mL CaCl2 1M + 3,8g glycerol qsp 20mL H2O and filter )
- Aliquote 500 muL in a CO2 ice and ethanol
Transformation with MH1 cells of the K1430379 biobrick from the 2010 plate
As some MH1 where unfrozen, we used a bit of them to transform a sensitive biobrick we have very few. The protocol followed is the usual heat-chock transformation protocol. The LB medium was intoduced at 11:30 today
