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.