Transformation DNA fragments (or plasmid DNA) into competent <I>E. coli</I>

Transformation DNA fragments (or plasmid DNA) into competent E. coli

* Caution: Use aerosol protecting tips if selection of transformed cells is not based on X-gal strategy.

  1. Remove competent cells (E.coli DH5aTM from GIBCO BRL) from -70 C freezer; thaw on wet ice.
  2. Place four 15-ml modified polystylene tubes (PST; Corning disposable sterile centrifuge tube) on ice.
  3. Gently mix cells (tapping with fingers), then aliquot 50ʵl competent cells into each of chilled the 15 ml PST.
  4. Add 1 µl of recombinant DNA sample (1 - 2 µg DNA) to the competent cells by moving the pipette through the cells while dispensing. Gently tap tubes to mix.
    * To prepare the recombinant DNA sample: if desired, wash the recombinant DNA of the interest (should be less than 10 kb which is usually digested with a restriction enzyme) twice in ultrafree 1.5 ml MFT by centrifugation (5000 rpm, 5 min, 4 C) by letting spin down to dead stop volume (ca. 5 to 20 µl).
    * Ultrafree MC polysulfone; 100,000 NMWL; Nihon Millipore Kogyo K.K. Yonezawa, Japan.
  5. Incubate cells on ice for 30 min.
  6. Heat-shock cells 45 sec in a 42 C water bath: Do NOT shake.
  7. Place on ice for 2 min
  8. Add 0.95 ml of room temperature SOC.
  9. Add 5, 10, 20, 50, 100, and 200 µl (duplicate) of the diluted DNA sample into 2.5 ml of 0.8 % LB at 42 C. Do this step and the next step within 5 min (-42 C).
    * Dilution of control DNA (pUC19) are duplicate of 5, 10, 50, 100, 200 µl of the diluted DNA samples. If size of the tranforming DNA is big size e.g. >5 Kb, start 50 µl diluted DNA sample into overlay medium.
  10. Do overlay on LB containng Carbenicillin plates (filter sterilized Crb 100 µg/ml or Amp 100 µg//ml).
  11. Incubate at 37 C.