CaCl2 Competent Cells

From 2010.igem.org

Revision as of 10:35, 24 August 2010

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.