Transformation DNA fragments (or plasmid DNA) into competent E. coli
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Transformation DNA fragments (or plasmid DNA) into competent E. coli
1.Remove competent cells (E.coli DH5aTM from GIBCO BRL) from -70 ℃ 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.
5.Incubate cells on ice for 30 min.
6.Heat-shock cells 45 sec in a 42 ℃ 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℃. Do this step and the next step within 5 min (-42℃).
10.Do overlay on LB containng Carbenicillin plates (filter sterilized Crb 100 µg/ml or Amp 100 µg//ml).
11.Incubate at 37℃.
