Preparation of Competent Cells and Transformation with pGEX DNA

In these procedures, E. coli host cells are made competent and then transformed with either uncut pGEX DNA or recombinant pGEX DNA.

If electroporation is used to transform the cells, see Appendix 3 (Electroporation). Otherwise, proceed as described below.

Transform 1 ng of uncut (supercoiled) vector DNA in parallel with recombinant pGEX ligations to determine the efficiency of each competent cell preparation.

This protocol is based on the procedure of Chung et al. (Chung, C. T. et al. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172–2175 [1989]).

All steps in this procedure should be carried out aseptically.

Reagents required

Use double-distilled water for preparation of all solutions.

Glycerol stock of E. coli host strain
LB medium and LB agar plates (prepared fresh): Combine 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 900 ml of water. Stir to dissolve, and adjust volume to 1 l. Sterilize by autoclaving. To prepare as a solid medium, add 1.2% to 1.5% agar.
TSS (transformation and storage solution) (ice-cold): For 100 ml: combine 1.0 g of tryptone, 0.5 g of yeast extract, 0.5 g of NaCl, 10.0 g of polyethylene glycol (Mr 3350), 5.0 ml of dimethylsulfoxide (DMSO), and 5.0 ml of MgCl2 (1 M) in 70 ml of sterile distilled water. Stir until dissolved. Adjust the pH to 6.5 with HCl or NaOH. Adjust to 100 ml with sterile distilled water. Sterilize by filtering through a 0.2 µm filter. Store at 4°C. Stable for up to 6 m.
LBG medium (LB + 20 mM glucose): Dissolve 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl in 900 ml of distilled water. Sterilize by autoclaving. After the medium has cooled to 50°C to 60°C, add 10 ml of sterile 2 M glucose. Adjust to 1 l with sterile distilled water. To prepare as a solid medium, add 1.2% to 1.5% agar.
LBGA medium and plates
(LBG + 100 µg/ml ampicillin):
See recipe for LBG medium, above. After autoclaving, cool the medium to 50°C, then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml). To prepare as a solid medium, add 1.2% to 1.5% agar.
Ampicillin stock solution: Dissolve 400 mg of the sodium salt of ampicillin in 4 ml of water. Sterilize by filtration and store in small aliquots at −20°C.
Glycerol: 80% in sterile distilled water
Procedure
  1. Using sterile technique, streak an E. coli host strain (e.g., JM109, BL21, etc.) from a glycerol stock onto an LB agar plate. Incubate overnight at 37°C.

  2. Isolate a single colony and inoculate 50 to 100 ml of LB broth. Incubate at 37°C with shaking at 250 rpm. Grow cells to an A600 of 0.4 to 0.5.

It is critical that the absorbance is not more than 0.5. This will take approximately 3 to 6 h.

Prewarming the broth to 37°C will shorten the growth time.

  1. Sediment the cells at approximately 2500 × g for 15 min at 4°C, then gently resuspend in 1/10 volume (5 to 10 ml) of ice-cold TSS and place on ice.

Cells must be used for transformations within 2 to 3 h.

Transformation of competent cells
  1. For each ligation reaction, as well as for the uncut vector control and the negative control (nontransformed competent E. coli host cells), add 1 ml of freshly prepared competent E. coli host cells to separate prechilled sterile disposable centrifuge tubes. Store on ice.

  2. Add 20 µl of each ligation reaction or 1 ng of uncut vector to the competent cells, swirl gently to mix, and place on ice for 45 min. Do not add any DNA to the negative control but instead add 20 µl of sterile distilled water.

  3. Incubate the tubes in a 42°C water bath for 2 min, then chill briefly on ice.

  4. For each sample, immediately transfer 100 µl of the transformed cells to a tube containing 900 µl of LBG medium (prewarmed to 37°C) and incubate for 1 h at 37°C with shaking (250 rpm).

  5. Plate 100 µl of the diluted, transformed cells from the ligated samples and 10 µl of the diluted, transformed cells from the uncut vector sample onto separate LBGA plates. Also plate 100 µl of the nontransformed, competent E. coli host cells. Incubate the plates at 37°C overnight, then proceed to screening.

  6. To prepare a frozen stock culture, add 100 µl of the diluted, transformed cells containing the pGEX DNA to 1 ml of LBGA medium and incubate for 30 min at 37°C with shaking at 250 rpm. After incubation, add 200 µl of sterile 80% glycerol and mix with a pipette tip. Store at −70°C.

Materials

     
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