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Transformation of DNA into Bacteria

Heat Shock, Electroporation and Blue/White Selection

Heat Shock

Items to have ready ahead of time:

  • LB media
  • 1.5-ml microfuge tubes
  • 42°C waterbath
  • Ice
  • 37°C waterbath and incubator
  • LB agarose plates with appropriate antibiotic

Protocol

  1. Thaw competent cells on ice, usually 100–200 µl per tube

    We most often use DH5α and XL-1 blue bacteria.
  2. Add a maximum of 20 µl of a ligation reaction, or 10-200 ng plasmid DNA.
  3. Mix very gently!
  4. Incubate the tubes on ice for 30 minutes.
  5. Heat shock the cells for 45 seconds at 42°C.

    The length of time may differ, depending on bacterial strain.
  6. Place the tubes immediately on ice for at least 2 minutes.
  7. Add 800 µl of LB medium (no antibiotics) to each tube.
  8. Incubate for 1 hour at 37°C, preferably shaking vigorously.
  9. Spin down briefly and remove most of the supernatant.
  10. Resuspend cell pellet with the 200 µl medium in the tube by pipetting.
  11. For ligations, plate the entire suspension on an LB plate containing antibiotic. For a plasmid transformation, plate out 10-20 µl on one LB plate and the remainder on a second plate containing the appropriate antibiotic.
  12. Incubate the plates overnight at 37°C.

Electro-Transformation

  1. Pre-chill cuvettes and LB or SOC media.
  2. Thaw competent cells on ice.

    Again, we most often use DH5α and XL-1 blue bacteria.
  3. Add 100 µl cells, plus 1 µl DNA solution, or no more than 1.5 µl of ligation mix, for electroporation, and electroporate at 2.5 kV/400 ohms (varies with size of cuvette).
  4. Immediately resuspend cells in chilled 800 µl LB and let recover at 37°C for 30 minutes.
  5. Plate on selective agar.

Blue/White Selection for XL-1 Blue Cells

  • Use this selection method when cloning into a vector (i.e., pBKS/pBSK), where you will disrupt a LacZ gene if an insertion occurs
  • Thirty minutes to 1 hour prior to spreading of transformed cells on plate, spread 100 µl 2% X-gal (in DMSO or dimethyl formamide) and 50 µl 0.1M IPTG (238 µg/ml H2O) on each plate.
  • The next day, colonies that have an insertion disrupting the LacZ gene will be white, and those with no insertion will be blue. You only need to screen the white colonies for proper insertion.

© 2014 The University of Texas MD Anderson Cancer Center