Liu 4/2004

E. coli competent cell protocol

Overview: There are several ways to prepare competent cells for plasmid DNA
transformation. This is the chemical method. Advantages are that its simple to
complete, requires no special equipment and gives good transformation efficiencies.
Disadvantages are that the efficiency is somewhat lower (vs. electroporation). In general,
it is best to use this when the transformation efficiencies is not the problem, otherwise
you might want to use and make the competent cells for electroporation.
Materials:
Single colony of E. coli cells to be transformed
LB medium
0.1 M CaCl2, ice cold
LB amp plates
42 C water bath
0.1 M CaCl2+15% glycerol, sterile
Procedure:
1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 C
overnight.
2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
Shake vigorously at 37 C to OD600 ~ 0.25-0.3 (usually it takes about 1.5-2 hours).
3. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M
CaCl2 plus 15% glycerol are on ice
4. Centrifuge the cells for 10 min at 3300 g (e.g. 4,000 rpm in the Jouan tabletop
centrifuge) at 4 C.
5. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2.
6. Keep the cells on ice for 30 min.
7. Centrifuge the cells as above.
8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution
plus 15% glycerol.
9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes.
Freeze these tubes on dry ice and then transfer them to -70 C freezer.
Notes:
1. The transformation efficiency is about 1-5x106/ul DNA when using the competent cells
prepared with this method.
2. Important: all steps after harvesting the cell should be done on ice (or at 4 C)
2. The frozen competent cells are stable for 6 months, but once a tube is taken from the
freezer and thawed, any unused portion should be discarded.
3. After the competent cells are made, the transformation efficiency should be checked
by transformation using plasmid DNA of known concentration.
Reference:
*Current Protocols in Molecular Biology (1.8.2)