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Star Republic: Guide for Biologists

Plasmid transformation

1. Remove competent cells (e.g. DH5a) from -70 C freezer, thaw on ice.

2. Gently mix thawed cells, aliquot 50 ml of competent cells into 1.5 ml eppendorf tubes.

3. Add 1 ml of DNA to the competent cells, mix by gentle tapping or pipetting.

4. Incubate on ice for 30 minutes.

5. Heat-shock cells in a 42 C degree water bath for 45 seconds.

6. Immediately put the cells back on ice for 5 minutes.

7. Add 1 ml of bacterial LB medium or SOC medium.

8. Incubate the cells in a 37 C shaker (~225 rpm) for 1 hour.

9. Meanwhile dry 1-4 LB plates with desired antibiotics in 37 C incubator. Invert the plate such that the cover is at the bottom.

10. Take an aliquot of cells (1-50 ml) and spread the cells on the plates. Or collect cells by brief centrifugation, remove LB media (leave ~ 50 ml). Re-suspend cells in the remaining media, and spread an aliquot on LB plates.

11. Grow cells overnight in a 37 C incubator. Plates should be inverted , i.e., covers at the bottom.

12. Don't overgrow cells at 37 C---cells don't contain plasmid (antibiotic resistant genes) may also form colony for prolonged incubation. Colony should be well-separated on the plates. If one is not sure of the DNA amount and/or transformation efficiency, several plates, each with different amount of cells spread on, should be used.

13. Plates can be stored at 4 C temporarily for a few days. For longer storage at 4 C, seal the plates with parafilm to prevent drying.