Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Making competent E. coli

7/95 from J. Kim

Day 1
1) Inoculate seed culture. Grow overnight at 37oC with shaking.

Day 2
2) Dilute the O.N. 1:100 in fresh medium.

3) Grow till A650=0.2-0.4.

4) Chill culture in ice water bath for 10 min.

5) Harvest cells at 5 K and 4oC for 10 min.

6) Resuspend in 1/4 culture volume 100 mM MgCl2; leave on ice for 5 min.

7) Spin at 4 K and 4oC for 10 min.

8) Resuspend in 1/4 culture volume 100 mM CaCl2; leave on ice for 20 min.

9) Spin at 4 K and 4oC for 10 min.

10) Resuspend in 1/20-1/40 culture volume of 100 mM CaCl2, 15% glycerol.

11) Make 50 ul aliquot into prechilled eppie tubes on ice; freeze in liquid nitrogen; store at -80oC.