Transformation Protocol

The following protocol explains how to transform chemically competent cells with DNA.

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  1. Thaw cells on wet ice after removing from -70?C.
  2. Mix cells gently by lightly flicking the tube. Aliquot ~50?l of cells into a chilled 1.5ml centrifuge tube(s). Unused cells may be refrozen, but a small drop in efficiency may result. For optimal recovery, refreeze cells in a dry ice/ ethanol bath prior to -70?C storage.
  3. Add DNA solution (?5?l per 50?l cells) to cell suspension and gently swirl tube(s) for a few seconds to mix. If a control is desired, repeat this step with 2?l of the provided pUC19 (50 pg/?l) in a separate tube.
  4. Incubate on ice for 30 minutes.
  5. Place tube(s) in 42?C water bath for ~30 to 45 seconds without shaking.

    Note: For the duration and temperature of this step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells? manufacturer.

  6. Place tube(s) again on ice for ~5 minutes.
  7. Dilute transformation reaction(s) to 1ml by addition of 950?l SOC medium provided.
  8. Shake tube(s) ~200 rpm for 60 minutes at 37?C.
  9. Plate by spreading 5-200?l of cell transformation mixture on LB agar plates containing the appropriate antibiotic and incubate overnight at 37?C.

When performing the pUC19 control transformation, plate 10?l of the transformation mixture on a LB agar plate containing 100?g/ml ampicillin. To facilitate cell spreading, place a pool of SOC (100?l) onto surface of plate prior to addition of transformation mixture.

Transformation Efficiency Calculation for Control DNA

For example:

If 40 colonies were obtained after transforming 100pg of pUC19 and plating 10?l of the final 1ml transformation mixture, the calculated transformation efficiency would be: