Transformation of Competent Cells
A. Preparing competent cells.
- Inoculate 30 ml of SOB broth in a 250-ml flask with bacteria to be transformed from a single colony on a fresh plate.
- Incubate overnight at 37°C with moderate agitation.
- Add 8 ml of the overnight culture to a 2-liter sidearm flask containing 200 ml of SOB broth. Incubate at 37°C with moderate agitation until an OD550 of approximately 0.3 is achieved.
- Collect the culture in four 50 ml sterile polypropylene centrifuge tubes, and chill on ice for 15 minutes.
- Pellet the cells by centrifugation at 3000 x g (5000 rpm using Sorval SS-34 rotor) for 15 minutes at 4°C. Drain the pellets thoroughly.
- Add 16 ml cold transformation buffer 1 (16 ml per 50 ml of initial culture). Resuspend the pellets by mild vortexing.
- Incubate tubes on ice for 15 minutes.
- Pellet the cells as before (3000 x g, 15 minutes, 4°C).
- Resuspend the pellets in a total of 16 ml of cold transformation buffer 2 (4 ml per 50 ml of initial culture). Store at 4°C no more than a few hours before use.
- Alternately, aliquot the cell suspension into 1.7-ml microcentrifuge tubes. Flash freeze by placing tubes in a dry ice/ethanol bath until frozen, and store at -70°C.
B. Transforming the cells.
- If competent cells have been stored frozen, thaw the tubes on ice.
- Pre-chill polypropylene tubes (Falcon 2059) on ice.
- Aliquot 300 µl of cells to the prechilled tubes.
- Add approximately 20 ul of plasmid DNA by gently stirring the cells while pipetting. Roll the tubes gently for a few minutes (on ice).
- Incubate the cells on ice for 40 minutes.
- Heat shock the cells by incubating at 42°C for 45 seconds. Do not shake cells!
- Add 1 ml of L-broth (no antibiotics) to each tube, and incubate cells at 37°C on a roller for 45 minutes to 1 hour to allow for plasmid expression.
- Plate out on appropriately supplemented solid media, and incubate transformants at 37°C overnight.
C. Media and buffers.
SOB Broth (1 liter)
Bacto tryptone 20.0 g Bacto yeast extract 5.0 g NaCl 0.6 g KCl 0.5 g MgCl2 10 mM (see below) MgSO4 10 mM (see below)
Note: SOB is identical to SOC, except that it contains no glucose.
Transformation Buffer 1 (500 ml)
- Dissolve tryptone, yeast extract, sodium chloride, and potassium chloride in a final volume of 990 ml distilled H2O. Sterilize by autoclaving.
- Just prior to using, add 10 ml of magnesium stock (see below) to the SOB broth to make the media 20 mM with respect to magnesium.
RbCl 6.0 g MnCl2·4H2O 5.0 g Potassium acetate 15.0 ml (1 M stock, pH 7.5) CaCl2·2H2O* 0.75 g Glycerol 75.0 ml Combine reagents in dH2O. Adjust pH to 5.8 with 0.2 M acetic acid. Bring to final volume of 500 ml with dH2O. Sterilize by filtration through a 0.22 µm disposable filter. Store at 4°C.
Transformation Buffer 2 (500 ml)
MOPS 10.0 ml (0.5 M stock, pH 6.8) RbCl 0.6 g CaCl2·2H2O* 5.5 g Glycerol 75.0 ml dH2O to 500.0 ml Combine reagents in dH2O. Bring to a final volume of 500 ml with dH2O. Sterilize by filtration through a 0.22 µm disposable filter. Store at 4°C.
* If using anhydrous CaCl2 use 0.57 g for Buffer 1, and 4.15 g for Buffer 2.
2 M Mg2+ stock (100 ml)
MgCl2 20.3 g MgSO4 24.7 g Dissolve reagents in a final volume of 100 ml dH2O. Sterilize by filtration through a 0.45 µm disposable filter. The resulting solution is 2 M with respect to Mg2+.
0.5 MOPS (100 ml)
Dissolve 10.47 g MOPS in dH2O. Adjust pH to 6.8 with NaOH. Bring to final volume of 100 ml. Sterilize by filtration through a 0.22 µm disposable filter. MOPS buffer should be clear and colorless.
1 M Potassium acetate (100 ml)
Disolve 9.82 g potassium acetate in dH2O. Adjust pH to 7.5 with acetic acid. Bring to final volume of 100 ml. Sterilize by autoclaving.
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Revised: Sunday, September 03, 1995 1:24:52 PM