Transformation Protocol for BL21(DE3) Competent Cells (C2527I)

New England Biolabs

Published: 2021-09-28 DOI: 10.17504/protocols.io.bgtxjwpn

Abstract

This transformation protocol is for the C2527I cells. (For the C2527H protocol, see here.)

Perform steps 2–9 in the tube provided.

Thaw a tube of BL21(DE3) Competent E. coli cells On ice until the last ice crystals disappear.

Mix gently and carefully pipette 50µL into a transformation tube On ice.

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.

Heat shock at exactly 42°C for exactly 0h 0m 10s. Do not mix.

Place 42On ice for 0h 5m 0s. Do not mix.

Pipette 950µL of 42Room temperature into the mixture.

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

10.

Warm selection plates to 37°C.

11.

Mix the cells thoroughly by flicking the tube and inverting.

12.

Perform several 10-fold serial dilutions in SOC.

13.

Spread 50µL–100µL of each dilution onto a selection plate and incubate 1h 0m 0s at 37°C.

Note

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