Team:Paris Bettencourt/Notebook/Drug Screening/Wednesday 17th July.html

Drug Screening

ASDF

Wednesday 17^th July

Making sD001+pD004 (Spect), sD001+pD005 (Chl31) competent

Making sD001+pD004 (Spect), sD001+pD005 (Chl31) competent, by using protocol 2 with these specifications:
The night before, cells were inoculated in a 5 ml culture and grown overnight with selection.
0.05 ml of the cell culture was diluted ~ 1:200 into 10 ml of selective media (Chl31,Spect) and grew to an OD600 of 0.6 – 0.7
(sD001+pD004 didn’t grow and protocol was continued for sD001+pD005 - see comment). Cells were Spun down at 4 ºC, 4000 rpm, 15 minutes.
Then were resuspended in 15 ml, ice-cold 100 mM CaCl2 and left for 3 hours.
Next, cells were spun down at 4 ºC, 4000 rpm, for 15 minutes, Resuspended in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol
and aliquoted into pre-chilled Eppendorf tubes. Tubes were immediately stored at -80ºC.
10 ml CaCl2 100nM + glycerol 15% was made from:

• 3.33 ml Glycerol 60% solution.
• 6.17 ml Sterile water
• 0.5 ml CaCl2 2 M solution.

Comment: sD001+pD004 did not grow overnight (and possibly always) in Streptavidin
instead of Spectinomycin so they most likely lost their resistance and we will make them again tomorrow by tranforming the BL21(DE3) cells with pD004.