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 � 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)

SOC , except that it contains no glucose.

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.

 

Transformation Buffer 1 (500 ml) RbCl 6.0 g MnCl2?H2O 5.0 g Potassium acetate 15.0 ml (1 M stock, pH 7.5) CaCl2?H2O* 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 � 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?H2O* 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 � 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 � 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 � 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.