Electroporation Protocol
New England Biolabs
Abstract
This protocol may be used with electrocompetent cells prepared according to this protocol.
Before start
For control electroporation dilute pUC19 to 10pg/μl with Milli-Q water.
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.
Steps
Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF .
Place recovery SOC in 37°C water bath.
Pre-warm LB-antibiotic plates at 37°C.
Thaw cells On ice for 0h 10m 0s or use freshly made cells.
Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes On ice.
Flick the tube containing cells a few times to mix and add 25µL to the microcentrifuge tubes.
Add 1µL to the cells in the microcentrifuge tube.
Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse ( don’t hold the button down ).
Immediately add 975µL, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
Rotate in the 37°C incubator for 1h 0m 0s.
Make appropriate dilutions.
Incubate at 37°C.
