Transforming Frozen Competent E. coli

Transforming Frozen Calcium Competent or SEM E. coli cells

1. Remove one tube of competent cells from -80°C freezer for each transformation. Let cells defrost on ice (~5-15 min).
2. Add the DNA (up to 10 µL for each 200 µL cells), flick the tube a few times to mix. Do NOT vortex the cells at this point.
3. Incubate for 30 min on ice.
4. Heat shock in water bath at 42°C during EXACTLY 45s.
5. Cool the tube for 2 min in a water/ice slurry for fast heat transfer.
6. Add pre-warmed liquid LB medium to one mL total volume, or use SOC or LB with 20 mM glucose (LB with 3.6 g/l glucose) and let cells recover at 37°C for 1 h. Put the tubes sideways in a shaking incubator if possible. Mixing is better in a 2 mL tube than in a 1.5 mL.
7. Centrifuge >12000g during 30 s or at lower speed (~5000 rpm for 2-3 min). Slower speed makes it easier to resuspend the cells.
8. Discard ~900 µL of the supernatant, do that 200-300 µL remains
9. Resuspend pellet with the remaining liquid.
10. Add the antibiotic if necessary, 1µL of a 1000x solution to for each ml of plate medium.
11. plate by adding 5-10 sterile glass beads and swirl the plate to spread the liquid.
12. Dry the plates opened in a Laminar air flow bench if necessary.
13. Incubate plates inverted at 37°C over-night. No parafilm is needed.