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Transformation of E. coli

Reference: H. Inoue, H. Nojima & H. Okayama, Gene 96, 23-28 (1990)

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.

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.

TB: 10 mM Pipes (or Hepes), 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl.
(Mix all components except for MnCl
2 and adjust pH to 6.7 with KOH. Then, dissolve MnCl2. Sterilize the solution by filtration through a prerinsed 0.45 um filter. Store the solution at 4 C.)

SOB: 2% Bacto-trypton, 0.5% Bact-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4
(Dissolve tryptone, yeast extract, NaCl and KCl in the purest water available. Autoclave for 30 min. Add 1/100 vol of filter-sterilized 1M MgCl2, 1M MgSO4.).

SOC: Add 1/100 vol of filter-sterilized 2M glucose to SOB.

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