Preparation of Competent Cells (from PMB)

1.Inoculate a couple of 20 ml LB broth in a 125 ml flasks with 0.5 ml of an overnight culture of the recipient strain.

2.Shake at 37 ℃ until O.D. ₆₀₀=0.13 - 0.15 (1 - 2 hours). Measure by taking some of the broth out with a sterile DPTP and reading in a plastic cuvette (discard cells afterward): If flanking arm flasks are accessible, use them for measuring O.D.

3.Spin the cells in sterile Oak Ridge Tube (5 K, 5 min, 4 ℃) and resuspend in 1 ml PMB #A. Then add another 9 ml PMB #A and spin the cells again (5 K, 5 min, 4 ℃).

4.Decant and resuspend in 1 ml PMB #B. Then add 9 ml more PMB #B.

5.Let sit on ice for 30 min, spin as above and resuspend in 2 ml PMB #B, add 0.2 ml glycerol and 30 µl DMSO. Use fresh or freeze in dry ice/EtOH.

PMB #A

1X 200 ml 1X

10 mM MOPS pH 7.0 210 mg Free acid

230 mg Na salt

10 mM Rubidium chloride 240 mg

Autoclave and store at 4 ℃

PMB #B

1X 200 ml 1X

10 mM MOPS pH 6.5 350 mg Free acid

85 mg Na salt

50 mM Calcium chloride 1.12 g

10 mM Rubidium chloride 240 mg

Autoclave and store at 4 ℃