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Star Republic: Guide for Biologists

Competent cells for transformation by electroporation

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1. Streak from a bacterial stock (e.g., DH5a, XL1-Blues) and grow cells on a LB plate over night in a 37 C incubator.

2. Pick a single colony and inoculate a 5 ml culture. Grow in a 37 C shaker (225 rpm) over night.

3. Add 1 ml of the over night culture to 200 ml LB media.

4. Grow cells until the OD600 reaches 0.5.

5. Chill bacteria on ice for 10 minutes and centrifuge at 3000 rpm for 15 minutes at 4 C.

6. Discard the supernatant, carefully resuspend the cell pellet in 200 ml sterile, ice-cold H2O.

7. Centrifuge and repeat step 6 once.

8. Centrifuge at 3000 rpm for 10 minutes at 4 C.

9. Discard the supernatant, carefully resuspend the cell pellet in 40 ml sterile, ice-cold 10% glycerol, mix.

10. Centrifuge at 3000 rpm for 10 minutes at 4 C and remove supernatant.

11. Estimate pellet volume, resuspend the cell pellet in equal volume of ice-cold 10% glycerol.

12. Aliquot 40-200 ml to sterile eppendorf tubes. Competent cells can be used immediately, or freeze at -70 C.