2) Grow cells at 37°C with vigrous shaking to an OD600 of 0.5 to 1.0 (the best results are
obtained with cells that grow rapidly; the appropriate cell density, therefore, depends
on the strain and growth condition).
3) To harvest, chill the flask on ice for 15 to 30 minutes, and spin in a cold rotor at
4000x g max for 5 minutes.
4) Remove as much of the supernatant as possible. Resuspend pellets in a total of 1 liter
of cold water. Spin as in step 3.
5) Resuspend in 0.5 liter of cold water. Spin as in step 3.
6) Resuspend in about 20 ml cold 10% glycerol. Spin as in step 3.
7) Resuspend in a final volume of 2 to 3 ml in cold 10% glycerol. The cell concentration
should be 1-3x1010 cells/ml.
8) The suspension could be frozened in aliquots on dry ice, and stored at -70°C. The
cells are good for at least 6 months under these conditions.
Electro-transformation
1) Gently thaw the cells at room temperature and place them on ice.
2) In a cold, 1.5 ml polypropylene tube, mix 40 µl of the cell suspension with 1-2 µl of
DNA (in either TE or water). Mix well and let sit on ice for about 1 minute.
3) Set the Gene Pulser apparatus at 25 uF and 2.5 kV. Set the Pulse Controller to 200
om.
4) Transfer the mixture of cells and DNA to a cold, 0.2 cm electroporation cuvette, and
shake the suspension to the bottom of the cuvette. Place the cuvette in a chilled safety
chamber slide. Push the slide into the chamber until the cuvette is seated between the
contacts in the base of the chamber.
5) Pulse once at the above settings. This should produce a pulse with a time constant of
4-5 msec. (The field strength will be 12.5 kV/cm.)
6) Remove the cuvette from the chamber and immediately add 1 ml of SOC medium to
the cuvette and quickly resuspend the cells with a pasteur pipette.
7) Transfer the cell suspension to a glass culture tube and incubate at 37°C for 1 hour
with shaking.
8) Plate on selective medium.
For 100 ml SOC medium;:
Take 94 ml LB medium which contains:
2% Bacto tryptone
0.5% Bacto yeast extract
10 mM NaCl
Add:
2.5 mM KCl 1 M 0.25 ml
10 mM MgCl2 1 M 1 ml
10 mM MgSO4 1 M 1 ml
20 mM glucose 10 % 3.57 ml
Ref: The Bio-rad Instruction Manaual.