Making your own electrocompetent cells
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0.5% yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
SOB + 20 mM glucose
Appropriate Antibiotics for Your ApplicationAntibiotics for Plasmid selection
Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).
Procedure (for 2, 250 ml cultures)
- Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm.
- Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks.
- Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
- Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
- Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.
- Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
- Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
- Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
- At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for >6 months.
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
- Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
- Place recovery SOC in 37°C water bath.
- Pre-warm LB-antibiotic plates at 37°C.
- Thaw cells on ice for 10 min or use freshly made cells.
- Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
- Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
- Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
- Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
- Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
- Rotate in the 37°C incubator for 1 h.
- Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:
Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
- Incubate overnight at 37°C.
If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)
cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)