Electrocompetent Cells Preparation Protocol for Lactobacillus Plantarum
- Inoculate 5ml MRS medium with L. plantarum freezer stock.
- Grow overnight at 30°C without shaking.
- Add 1.25g glycine to two flasks of 50ml MRS medium.
- Shake to dissolve.
- Add 1ml overnight culture to each flask (1:50 dlution).
- Culture for ~3 hours at 37°C with shaking.
- Centrifuge culture for 5min at 4000g
- Resuspend in 25ml ice-cold DI water.
- Repeat it.
- Centrifuge for 5min at 4000g
- Resuspend in 5ml 50mM EDTA.
- Incubate on ice for 5 minutes
- Add 25ml ice-cold DI water.
- Centrifuge culture for 5min at 4000g
- Resuspend in 25ml ice-cold DI water.
- Centrifuge culture for 5min at 4000g
- Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
- Repeat it.
- Centrifuge culture for 5min at 4000g
- Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
- Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
- Keep on ice until use (within the next two hours).
- Add 10μL of plasmid DNA to the 90μL of cell concentrate.
- Keep on ice for 5 minutes.
- Put 1mm cuvettes on ice too.
- Pipette the cell/DNA mixture into the cuvette
- Electrporate at 1200 volts
- Time constant should be ~5.0
- Immediately transfer the electroporated cells to 900μL of MRS medium.
- Incubate at 30°C for 2-3 hours.
- Plate on medium with the appropriate antibiotic
- Incubate at 30°C for two days.
- Pick colony
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