Home Protocols Transformation Protocol (NEB #E8201)

Transformation Protocol (NEB #E8201)

  1. Thaw chemically competent NEBExpress cells (NEB #C2523) on ice.

  2. Add 2 μl of the chilled assembled product to the competent cells. Mix gently by pipetting up and down. Do not vortex.

  3. Place the mixture on ice for 30 minutes. Do not mix.

  4. Heat shock at 42°C for 30 seconds. Do not mix.

  5. Transfer tubes to ice for 2 minutes.

  6. Add 950 μl of room-temperature SOC media to the tube.

  7. Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

  8. Warm LB plates containing 100 μg/ml ampicillin to 37°C.

  9. Spread 100 μl of the cells onto the LB ampicillin plates. Do not plate on IPTG.

  10. Incubate overnight at 37°C.

  11. Screen for the presence of inserts in one or more of the following ways:

  1. Perform colony PCR on several transformants using appropriate primers (see Appendix C item 1.2, p. 11 in the manual).

  2. Prepare miniprep DNA. Digest with an appropriate restriction endonuclease to determine the presence and orientation of the insert.

  3. SDS-PAGE analysis of transformed cells:

  1. Inoculate several transformants into 5 ml of LB containing 100 μg/ml ampicillin and grow to 2 x 108 cells/ml (OD600 of ~ 0.5).

  2. Split each sample into two 2.5 ml cultures.

  3. Add IPTG to one of the cultures to a final concentration of 0.3 mM (for example add 7.5 μl of a 0.1 M IPTG stock solution.) Incubate at 37°C with good aeration for 2 hours.

  4. Withdraw a 0.5 ml sample from each culture. Microcentrifuge for 1 minute, discard the supernatant and resuspend the cells in 100 μl SDS-PAGE sample buffer (NEB #B7703).

  5. Place samples in a boiling water bath for 5 minutes. Analyze 10 μl of each sample by SDS-PAGE along with a protein standard (NEB #P7717) and 15 μl of the supplied MBP6 in SDS-PAGE Sample Buffer. Stain the gel with Coomassie brilliant blue.

For pMAL-c6T constructs, an induced band should be visible at a position corresponding to the molecular weight of the fusion protein. A band at or around the position of MBP6 (MW 45.5 kDa) indicates either an out of frame fusion or a severe protein degradation problem. These can usually be distinguished by performing a Western blot using the Anti-MBP Monoclonal Antibody; even with severe protein degradation, a full-length fusion protein can usually be detected on the Western.