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
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.
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 | pH 6.7 | |
| pH 6.7 - 7.0 |
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