Transformation Ultra-Competent E. coli (Inoue Method)

Wednesday, November 19, 2003

Description
Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28.

Procedure
1. Inoculate from an overnight grown in LB.
2. Grow in 250 ml "SOB" at 18C until OD600 = 0.6.

2. On ice for 10 minutes.

3. Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.

4. Resuspend cells gently in 80 ml of ice cold "TB". 5. On ice for 10 minutes.

6. Spin at 2500 x g (5000 rpm in a Sorvall GSA, 5500 rpm in a Sorvall SS-34, or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.

7. Resuspend cells gently in 20 ml of ice cold "TB".

8. Add DMSO to a final concentration of 7%.

9. Place on ice for 10 minutes.

10. Aliquot into 1-2 ml and freeze in liquid nitrogen.

11. Store in liquid nitrogen.

Recipes
SOB Medium and TB (Transformation Buffer)
SOB 2% (w/v) bacto tryptone TB
0.5% (w/v) yeast extract 10 mM Pipes
10 mM NaCl 55 mM MnCl2
2.5 mM KCl 15 mM CaCl2
10 mM MgCl2 250 mM KCl
10 mM MgSO4 adjust pH to 6.7 with 5N KOH prior to adding the MnCl2

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Transformation Ultra-Competent E. coli (Inoue Method)