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1. Culture E. coli in 250 ml of SOB medium in a 2-liter flask at 18 C with vigorous shaking until an A260 of 0.6 is reached.TB: 10 mM Pipes (or Hepes), 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl.2. Keep on ice for 10 min.
3. Spin at 3000xg (4000 rpm in Hitachi RPR-10 rotor) for 10 min at 4 C.
4. Resuspend the pellet in 80 ml of ice-cold TB. Keep in an ice-water bath for 10 min.
5. Spin as above. Resuspend the pellet in 20 ml of TB. Add DMSO with gentle swirling to a final concentration of 7%. Keep the centrifugation bottle in ice-water bath for 10 min.
6. Dispense by 1-2 ml into microfuge tubes. Freeze immediately in liquid nitrogen.@Store the frozen competent cell at -80C.
7. Thaw the competent cell at room temperature. Dispense by 200 ul into microfuge tubes on ice.
8. Add plasmid solution (<5 ul). Keep on ice for 30 min.
9. Heat at 42 C for 30 seconds without agitation. Transfer onto ice.
10. Add 0.8 ml of SOC. Incubate at 37 C for 1 hr.
11. Streak on plates containing appropriate antibiotics. Incubate the plates overnight at 37 C.
SOB: 2% Bacto-trypton, 0.5% Bact-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl
2, 10 mM MgSO4SOC: Add 1/100 vol of filter-sterilized 2M glucose to SOB.
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