Transformation is the process of getting the recombinant vector (one containing the gene of interest) from a reaction mixture into cells, such as E. coli BL21(DE3)pLysS cells. To enable the cells to take up circular vector DNA they have to be made competent. The method for preparation of competent cells depends on the transformation method used and transformation efficiency required. For a high transformation efficiency transformation, we use electroporation and electrocompetent cells.
Electroporation of E. coli BL21(DE3)pLysS Cells
- 10% glycerol (made with deionised water). Autoclave and then refrigerate (at 4°C) until required.
- LB broth (200 ml), autoclaved.
- SOB broth, autoclaved.
- 2 M glucose: Make up in deionised water and filter-sterilise using 0.22 μm filters. Keep at 20°C in aliquots.
- 1 M MgCl2: Make up in deionised water and filter-sterilise using 0.22 μm filters. Keep at room temperature
- 1 M MgSO4: Make up in deionised water and filter-sterilise using 0.22 μm filters. Keep at room temperature.
- 250 mM KCl: Make up in deionised water and filter-sterilise using 0.22 μm filters. Keep at room temperature.
- Antibiotics [either kanamycin or ampicillin]: Kanamycin stock solutions are 10 mg/ml in deionised water, kept in aliquots at 20°C.
- Chloramphenicol: Stock solution is 34 mg/ml made up in 100% ethanol and aliquots are stored at 20°C.
- Liquid nitrogen.
- SOC broth. To 9.6 ml SOB, add 100 μl each of 2M glucose, 1M MgCl2, 1M MgSO4 and 250 mM KCl. Mix well, keep on ice and use within 1 hour.
Hardware and consumables required
- Bio-Rad MicroPulser Electroporation Apparatus. This lives in Hugh Patterton’s lab on the 4 th floor of MCB. It comprises of a pulse generator module and a shocking chamber.
- Electroporation cuvettes. Blommie keeps these. Try and get ones with 0.1 cm gaps instead of 0.2 cm.
- Ice-box filled with ice
- Sterile tips and Eppendorf tubes
- Fresh LB agar plates that have been dried in an oven at 50°C overnight.
- Beckman centrifuges, precooled. Use JA-14 rotor with tubes. Set 4 000 x g as 5105 RPM.
1. Preparation of electrocompetent E. coli BL21(DE3)pLysS cells
- Grow up a 5 ml overnight culture at 30°C with 34 μg/ml chloramphenicol with vigorous shaking.
- Inoculate 200 ml of fresh LB (with 34 μg/ml chloramphenicol) with 500 μl of the overnight culture and grow at 37°C for around 5 hours.
- The best results are obtained with cells that are harvested at early to mid-log phase (OD600 ~ 0.2-0.5).
- Chill cells on ice for at least 30 min. For all subsequent steps, keep the cells as close to 0°C as possible. To harvest, transfer the cells to a cold centrifuge tube and spin at 4000 x g for 15 min at 4°C.
- Carefully pour off and discard the supernatant. It’s better to sacrifice the yield by pouring off a few cells than to leave any supernatant behind.
- Gently resuspend the pellet in 200 ml of ice-cold 10% glycerol. Centrifuge at 4 000 x g for 15 min at 4°C. Carefully pour off the supernatant and discard the supernatant.
- Gently resuspend the pellet in 100 ml of ice-cold 10% glycerol. Centrifuge at 4 000 x g for 15 min at 4°C. Carefully pour off the supernatant and discard the supernatant.
- Gently resuspend the pellet in 8 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min at 4°C. Carefully pour off the supernatant and discard the supernatant.
- Resuspend the cell pellet with 400 μl of 10% glycerol. Aliquot 40 μl volumes into pre-chilled Eppendorf tubes and quickly freeze in liquid nitrogen. Store the aliquots at 70°C. I have not assessed how long electro-competent BL21 cells are stable at this temperature. I usually use all the aliquots within 1 week.
- Thaw the frozen electro-competent BL21 cells on ice. For each sample to be electroporated, place a 0.1 cm electroporation curette on ice.
- Ensure that the plasmid DNA to be electroporated is salt-free and that any ligase has been heat-inactivated. Add 1-2 μl plasmid DNA to the 40 μl BL21 aliquot and leave on ice for ~ 1 minute.
- Set the Micropulser to Manual and choose a potential difference of 1.0 kV.
- Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom of the gap. Place the cuvette in the chamber slide. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. Press the pulse button once only.
- Remove the cuvette from the chamber and IMMEDIATELY add 1 ml of SOC medium to the cuvette. Quickly, but gently resuspend the cells with a micropipette or sterile Pasteur pipette. The period between applying the pulse and transferring the cells to outgrowth medium is crucial for recovering E. coli transformants. Delaying this transfer by even 1 min causes a 3-fold drop in transformation.
- Record the pulse parameters. The time constant should be 5 milliseconds or greater.
- Transfer the cell suspension to a sterile Eppendorf tube and incubate at 37°C for 2 hours with shaking.
- For selecting transformants, plate 100 μL on LB agar containing the appropriate antibiotics and grow overnight at 37°C.