Team:Groningen/Protocols

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CaCl2 Competent Cells

Day 1:

-Inoculate 5 ml LB( TY) medium with E.coli from glycerol stock

-Grow overnight at 37 oC

Day 2:

-Inoculate 20 ml LB medium with 200 μl overnight culture

-Grow at 37 oC until OD600 = 0,3-0,5(+/- 2 hours)

-Spin down 5 minute at 4000 rpm at 4 oC

-Resuspend in 10 ml chilled 0,1 M caCl2 (from here, keep on ice! )

-Inoculate on ice for 20 minutes

-Spin down as before

-Remove supernatant

-Resuspend in 2 ml 0,1 M CaCl2 /10% glycerol

-Divide 100 μl aliquots

-Store competent cells in - 80 oC