CaCl2 Competent Cells

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-'''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
-'''Transformation protocol'''
-Melt agar in autoclave or microwave. Cool down to 60 oC. One jar ( 100mL ) is enough for 7 plates.
--Add 10 μl of ligation product to 100 μl of cells ( don't for get controls! )
--Incubate 30 min on ice (make agar plates)
--Incubate for 1 min at 42 oC
--Add 400 μl of TY
--Incubate for 30 min at 37 oC (dry agar plates)
--Plate out. Use 250 μl of the transformed cells.
-*If you expect the transformation to be very efficient (for instance using undigested plasmid DNA) first make dilution (1:10 or 1:100 ) and plate 250 μl of this
-*If you expect the trasformation to be inefficient, concentrate cells by spinning the 500 μl and resuspending the pellet in 250 μl, which you will use for plating.

Latest revision as of 14:10, 24 August 2010