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 ℃ |
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