z TSS method for competent cell preparation

Reagent:TSS solution

85% LB medium

10%PEG(wt/vol,MW8000)

5%DMSO(vol/vol)

50mM MgCl2(PH6.5)

Autoclave or filter sterilize.Storage at 4℃for 2 weeks.

1.Streak the cell stock on a LB plate.Incubate the plate at 37℃ overnight.

2.Pick a signle ,well –isolate colony and incubate it into 5ml of LB

broth.Incubate at 37℃overnight with shaking at 220rpm.

3.Transfer 1 ml of the saturated overnight culture to a a sterile 500-ml flask

containing 100ml of LB medium .Incubate the cell at 37℃ with the

shaking at 220 rpm,until OD600 reach 0.5,this usually takes 2.5-3.0 hr.

4.Then chill the flask on the ice fir 20 mins and then collect the cells by

centrifugation at 3000 rpm for 5 min at 4℃.

5.Resuspend the cells in 10 ml of ice-cold TSS solution.Now the competent

cells are ready to be transformed. Please do this for two times.

6.Aliquot 150μl competent cells to 1.5 ml tube.Storage for a long time at -70

℃.

z KCM method for transforming

KCM solution:

5×KCM= 100ml 10ml

0.5M KCl 3.73g 2.5ml(2MKCl)

0.15M CaCl

2 2.21g 2.5ml(1M MgCl

2

)

0.25M MgCl

2 5.08g 1.5ml(1M CaCl

2

)

H2O up to 10 ml

1.Slow thaw an aliquot of cells on ice.

2.Set up a second tube containing 20μl of 5×KCM ;Add DNA and H2O up to a

total volume of 100μl.Keep on ice.

3.Add 100μl of the cell suspension to the second tube;mix and keep on ice for

20 mins.

4.Heat shock the cells by transferring to 42 ℃ for 90s.

5.Add 1 ml of pre-warmed LB medium to the tube;incubate at 37℃ for

40-60minutes.

6.Plate out 100μl of the transformation onto selective medium.Spin down the

remaining cells,resuspend into 100μl of fresh LB and plate onto a second plate.下载本文