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1 ng of uncut plasmid DNA typically produces 2-5 X 10^3 drug resistant colonies when HB101, DH5, or NM522 cells are transformed using this procedure. GM161 (dam-) and GM119 (dam-, dcm-) cells are ca. 50-fold less efficient. Do not add more than 10 uL DNA to 100 uL of cells.


  1. Materilas

    1. 2X YT broth
      15g tryptone
      10g yeast extract
      5g NaCl
      1liter H2O
      For plates add 15g agar.

  2. Methods

    1. Thaw 100 uL aliquot of concentrated, CaCl2 treated E. coli cells on ice.
    2. Add DNA and mix well by tapping tube with finger.
    3. Incubate on ice at least 30 minutes with occasional mixing as above.
    4. "Heat-shock" for 90 seconds at 42C (37C works almost as well).
    5. Incubate on ice at least 2 minutes.
    6. Add 1 ml of 2X YT broth, mix gently, and incubate at 37C for 45-60 minutes (no aggitation).
    7. Spread 10-100 uL on 2X YT plates2 containing the appropriate antibiotic at the recommended concentration and incubate them at 37C overnight.

D. S. Waugh, 06/07/88

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