High Efficiency Transformation Protocol (C2987H)

New England Biolabs

Published: 2022-02-16 DOI: 10.17504/protocols.io.bddti26n

Abstract

This is the protocol for C2987H cells. If you are using the C2987I cells, please refer to this protocol.

Before start

For this protocol, perform steps 1-8 in the tube provided.

Steps

Thaw a tube of NEB 5-alpha Competent E. coli cells On ice for 0h 10m 0s.

Note

Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.

Add 1µL-5µL containing 1pg-100ng to the cell mixture.

Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

Place the mixture On ice for 0h 30m 0s. Do not mix.

Note

For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.

Heat shock at exactly 42°C for exactly 0h 0m 30s. Do not mix.

Note

Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.

Place On ice for 0h 5m 0s. Do not mix.

Pipette 950µL into the mixture.

Place at 37°C for 1h 0m 0s, shaking vigorously (250rpm,0h 0m 0s) or rotating.

Note

Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

Warm selection plates to 37°C.

Note

Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

10.

Mix the cells thoroughly by flicking the tube and inverting.

11.

Perform several 10-fold serial dilutions in SOC.

12.

Spread 50µL-100µL of each dilution onto a selection plate.

13.

Incubate 1h 0m 0s at 37°C.

Note

Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48h 0m 0s.