Koshland Web Site/Methods


(Inoue et al., 1990 Gene 96:23)
  1. Inoculate a 5ml overnight of E.coli in LB+20 mM MgSO4.
  2. Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an A600 of 0.4-0.6. Temp is important--see original ref.
  3. Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.
  4. Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.
  5. Spin cells 3K, 10', 4°C.
  6. Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.
  7. Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106 cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.

To use:

  1. To 50 or 100 ul cells, add 5-50 ng DNA. Leave 30’ on ice.
  2. Heat shock, 45 sec at 42C, then chill cells on ice about 2’.
  3. Spin down (15 sec, eppendorf fuge) and remove SN. (Removing the Manganese seems to boost efficiency about 10X) and resuspend cells in 200 ul LB.
  4. For a supercoiled plasmid, plate 1 ul of cells. For a ligation, plate 20 ul and the rest.

TB (transformation buffer: filter sterilize and store 4°C)

Product [stock] [ ]final volumes to make 100ml
Pipes-NaOH pH6.7 0.5M 10 mM 2 ml
CaCl2 0.5M 15 mM 3 ml
KCl 2M 0.25M 12.5 ml (or 1.864g)
MnCl2 1M 55 mM 5.5ml (or 1.088g) add to 100ml with ddH2O
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