CaCl2 Competent Cells

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Revision as of 11:23, 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

-Incubate cells on ice for 2 min

-Add 400 μl of SOC medium

-Incubate for 30 min at 37 oC on shaker

-Spread 100-300 μl onto a plate made with appropriate antibiotic

-Grow overnight at 37 °C.

-Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.

• 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.

Growing streptomyces