Electroporation Protocol (C2986)
New England Biolabs
Abstract
NEB Turbo Electrocompetent E. coli cells are suitable for high efficiency electroporation and rapid colony growth. These cells are ideal for DNA library constructions and all cloning purposes.
Before start
Steps
Preparation
Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. VWR #60818-667) at Room temperature.
Place SOC recovery medium in a 37°C water bath.
Pre-warm selective plates at 37°C for 1h 0m 0s.
Electroporation
Place electroporation cuvettes (1 mm) and microcentrifuge tubes On ice.
As a positive control for transformation, dilute the control pUC19 by 1:5 to a final concentration of 10pg/μl using sterile water.
Thaw NEB Turbo Electrocompetent cells 37On ice (about 0h 10m 0s ) and mix cells by flicking gently.
Transfer 25µL (or the amount specified for the cuvettes) to a chilled microcentrifuge tube.
Add 1µL.
Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette.
Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators:
2.1 kV, 100 Ω, and 25 μF
The typical time constant is ~2.6 milliseconds.
Immediately add 975µL to the cuvette.
Gently mix up and down twice .
Transfer to the 17 mm x 100 mm round-bottom culture tube.
Shake vigorously (250rpm,0h 0m 0s) or rotate at 37°C for 1h 0m 0s.
Dilute the cells as appropriate, then spread 100µL-200µL onto a pre-warmed selective plate.
Incubate plates 8h 0m 0s to at 37°C.
