Transformation Ultra-Competent E. coli

 

Transformation "Ultra-Competent" E. coli (Inoue Method)

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

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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.

 

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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 thanks to Markus Schneemann for the tip!
pH 6.7 - 7.0