Transformation via Electroporation

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Purpose

This protocol is used to transform electrocompetent cells with a plasmid.

The electrocompetent cells are made using the protocol found on Preparation of Competent Cells for Electroporation.

Materials Needed

-Electroporation apparatus

-1 mm electroporation cuvette

-micropipette

-LB broth

-Electrocompetent cells

-DNA

-microfuge tubes

-ice

-kimwipes

-LB or NBY agar plates

-28 C incubator

Protocol

-Set the electroporation apparatus to 1.8 kV, 25 microfarads, and the pulse controller to 200 omega

-Add 1.5 microliters of DNA to be transformed to 40 microliters of electrocompetent cells on ice and mix

-Transfer the DNA and cells to a pre-chilled 1 mm electroporation cuvette

-Wipe the cuvette with a kimwipe and place it into the sample chamber

-Deliver a pulse by pushing in charge button

-Remove the cuvette and ad 1 mL LB broth to the cuvette

-Transfer the broth and cells to a sterile microfuge tube and incubate for 2 hours at 28 C with shaking

-Place aliquots of the mixture on LB or NBY agar plates with the appropriate antibiotics for selection the cells

-Incubate at 28 C for 2 days

References

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