How to Perform a Transformation with NEB Competent Cells

Use this protocol for maximum transformation efficiency.

Script

The following transformation protocol is recommended for use with New England Biolabs’ competent cells.

Note: If using competent cells from another supplier, we suggest following their recommended protocols for optimum transformation efficiency.

1. Thaw one tube of competent cells on ice.

2. Place DNA on ice.

3. Add DNA to 50 µl of competent cells. Mix gently by pipetting up and down or flicking the tube 4 to 5 times to mix the cells and DNA. Do not vortex.

4. Incubate the mixture on ice for 30 minutes. Do not mix.

5. Heat shock the mixture at 42°C for 10 to 30 seconds depending on strain. Do not mix.

6. Incubate on ice for 5 minutes.

7. Add 950 µl of room-temperature SOC media to the transformation mixture.

8. Incubate the transformation mixture at 37°C for 60 minutes. Shake vigorously at 250 rpm, or rotate.

9. Warm selection plates to 37°C.  Spread 50 to 100 µl of the transformation mix onto the plates. Many scientists choose to plate three different outgrowth concentrations to ensure optimal colony counts.

10. Incubate the plates overnight at 37°C. You should have colonies on your plate the next morning.

The mechanism of transformation can be found in an animation located at www.neb.com/transformationanimation. neb.com contains many helpful resources to help select the best competent cell strain for your needs, including NEBcloner. Just answer a few simple questions and the tool will indicate which strain will work best for you.

Loading Spinner