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. Add 5-10 sterile glass beads to the plate
8. If you expect lots of transformants, add 200 µL of cells to the middle of a plate and proceed to plating below. Save the remaining cells on the bench. If you need high efficiency concentrate transformants like below.

Concentrate transformants

1. Centrifuge >12000 g during 30 s or at lower speed (~5000 rpm for 2-3 min). Slower speed makes it easier to resuspend the cells.
2. Make sure cells have settled to a pellet. Discard ~900 µL of the supernatant, so that 200-300 µL remains.
3. Resuspend pellet with the remaining liquid with a pipette.
4. Add all of the cells to the the middle of a plate.

Plating

1. Add 20 µL of a 1000 x antibiotic to the puddle with cells. Do this rapidly so that cells do no
2. Plate by and swirl the plate to spread the liquid.
3. Dry the plates opened in a Laminar air flow bench if necessary.
4. Incubate plates inverted at 37°C over-night. No parafilm is needed.

Reference: Inoue, H, H Nojima, and H Okayama. 1990. “High Efficiency Transformation of Escherichia Coli with Plasmids.” Gene 96 (1): 23–28. https://doi.org/10.1016/0378-1119(90)90336-p.