实验概要

        Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the bacteria extensively at low temperature, and then resuspending them in a solution of low ionic strength containing glycerol. DNA is introduced during exposure of the bacteria to a short high-voltage electrical discharge.

实验材料

Buffers and Solutions

        Glycerol (10% v/v) (molecular biology grade), ice cold

        Pure H2O

        Milli-Q or equivalent, sterilized by filtration through prerinsed 0.45-μm filters. Store at 4°C.

Nucleic Acids

        Plasmid DNA (recombinant plasmid)

Media

        GYT medium, ice cold

        LB medium, prewarmed to 37°C

        SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic

        SOC medium

实验步骤

1. Inoculate a single colony of E. coli from a fresh agar plate into a flask containing 50 ml of LB medium. Incubate the culture overnight at 37°C with vigorous aeration (250 rpm in a rotary shaker).

2. Inoculate two aliquots of 500 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/minute in a rotary shaker). Measure the OD600 of the growing bacterial cultures every 20 minutes.

3. When the OD600 of the cultures reaches 0.4, rapidly transfer the flasks to an ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in an ice-water bath.

4. Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 500 ml of ice-cold pure H2O.

5. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 250 ml of ice-cold 10% glycerol.

6. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Decant the supernatant and resuspend the pellet in 10 ml of ice-cold 10% glycerol.

7. Harvest cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Carefully decant the supernatant and use a Pasteur pipette attached to a vacuum line to remove any remaining drops of buffer. Resuspend the pellet in 1 ml of ice-cold GYT medium.

8. Measure the OD600 of a 1:100 dilution of the cell suspension. Dilute the cell suspension to a concentration of 2 x 10¹⁰ to 3 x 10¹⁰ cells/ml (1.0 OD600 = approx. 2.5 x 108 cells/ml) with ice-cold GYT medium.

9. Transfer 40 μl of the suspension to an ice-cold electroporation cuvette (0.2-cm gap) and test whether arcing occurs when an electrical discharge is applied (please see Step 16 below). If so, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5mEq).

10. To use the electrocompetent cells immediately, proceed directly to Step 12. Otherwise, store the cells at -70°C until required. For storage, dispense 40-μl aliquots of the cell suspension into sterile, ice-cold 0.5-ml microfuge tubes, drop into a bath of liquid nitrogen, and transfer to a -70°C freezer.

11. To use frozen electrocompetent cells, remove an appropriate number of aliquots of cells from the -70°C freezer. Store the tubes at room temperature until the bacterial suspensions are thawed and then transfer the tubes to an ice bath.

12. Pipette 40 μl of the freshly made (or thawed) electrocompetent cells into ice-cold sterile 0.5-ml microfuge tubes. Place the cells on ice, together with an appropriate number of bacterial electroporation cuvettes.

13. Add 10 pg to 25 ng of the DNA to be electroporated in a volume of 1-2 μl to each microfuge tube and incubate the tube on ice for 30-60 seconds. Include all of the appropriate positive and negative controls.

14. Set the electroporation apparatus to deliver an electrical pulse of 25 μF capacitance, 2.5 kV, and 200 ohm resistance.

15. Pipette the DNA/cell mixture into a cold electroporation cuvette. Tap the solution to ensure that the suspension of bacteria and DNA sits at the bottom of the cuvette. Dry condensation and moisture from the outside of the cuvette.Place the cuvette in the electroporation device.

16. Deliver a pulse of electricity to the cells at the settings indicated above. A time constant of 4-5 milliseconds with a field strength of 12.5 kV/cm should register on the machine.

17. As quickly as possible after the pulse, remove the electroporation cuvette and add 1 ml of SOC medium at room temperature.

18. Transfer the cells to a 17 x 100-mm or 17 x 150-mm polypropylene tube and incubate the cultures with gentle rotation for 1 hour at 37°C.

19. Plate different volumes (up to 200 μl per 90-mm plate) of the electroporated cells onto SOB agar medium containing 20mM MgSO4 and the appropriate antibiotic.

20. Store the plates at room temperature until the liquid has been absorbed.

21. Invert the plates and incubate them at 37°C. Transformed colonies should appear in 12-16 hours.