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

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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 A 260 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 CaCl 2 , 250 mM KCl.(Mix all components except for MnCl 2 and adjust pH to 6.7 with KOH. Then, dissolve MnCl 2 . 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 MgCl 2 , 10 mM MgSO 4 (Dissolve tryptone, yeast extract, NaCl and KCl in the purest water available. Autoclave for 30 min. Add 1/100 vol of filter-sterilized 1M MgCl 2 , 1M MgSO 4 .).

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

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