1.                  Thaw competent cells on ice. It is essential that cells be completely thawed before pipetting. When cells are completely thawed, you should see clearly two phases: the upper clear phase and the bottom cloudy phase. Pipet slowly and don’t pipet cells up and down.

2.                  Remove bacterial plates from refrigerator and put pre-determined number of plates with the appropriate antibiotic into a 37^oC incubator.

3.                  Transfer 100 or 50 ml of cells to a prechilled Falcon 352059 tube or an equivalent tube, add DNA of your choice (pure DNA or ligation mix) to the cells and incubate on ice for 30 minutes (may use 5 min for subcloning). Heat-shock at 42°C for 30 seconds and put the tube on ice for 2 minutes.

4.                  Add 900 or 950 ml SOC, depending on whether 100 ml or 50 ml cells are used, and shake the cells at 37°C at 150 rpm for 60 minutes.

5.                  Plate out desired amount on a prewarmed (37°C) plate with the appropriate antibiotic and incubate in a 37^oC incubator.

[ Home ] [ Up ]

Last modified: 04/25/06