Kitto Lab, The University of Texas at Austin
http://research.cm.utexas.edu/bkitto/Kittolabpage/Protocols/Microbiology/electroporation.html
This procedure prepares glycerol stock cultures of bacteria for electroporative transformation.
Preparation of Electrocompetent Cells
Materials
- LB Broth Base [Life Technologies, cat # 12795-027]: add 25.0 g per liter of dH2O; autoclave at 121 °C for at least 15 minutes. Store at 4 °C.
- 10% Glycerol: autoclave at 121°C for at least 15 minutes, store at 4 °C.
- Bacterium to be grown (plate, culture, or stock)
- 37°C Shaker/incubator
- 25 mL LB in 125 mL flask (autoclaved)
- 250 mL LB in 1000 mL flask (autoclaved)
Procedure
- Add colony of desired bacterium to 25 mL LB flask; grow overnight.
- Add 1 mL from overnight culture to 250 mL LB flask; grow until bacteria are in log phase (4-5 hours for E. coli)
- Split culture across two centrifuge flasks
- Centrifuge at 2600g for 15 min in a 4 °C superspeed centrifuge
- In cold room or on ice, discard supernatant and resuspend cell pellets in 2 x 125 mL 10% glycerol. FROM HERE ON, ALWAYS KEEP CELLS COLD!!!
- Centrifuge as before; discard supernatant and resuspend as before; recentrifuge and discard supernatant
- In cold room or on ice, suspend pelleted cells in 2 x 1 mL 10% glycerol; combine fractions and split into 100 µL aliquots
- If you are not going to use them immediately, quick-freeze in liquid nitrogen; store at -80 °C
Electroporation
Materials
- Life-Technologies Cell Porator
- Ice
- 1 mL/sample of rich media such as SOC for your cells ro recover in.
- Electroporation chambers (cuvettes)
- Growth plates with proper antibiotic for plasmid.
- Plating rod
- 37 °C shaking incubator and regular incubator.
Procedure
- Be sure power is off on the voltage booster and pulse control apparati. If you are not carefull, serious injury could result.
- Fill the chamber safe with an ice-water slurry. Place the chamber rack in the chamber safe.
- Label electroparation chambers (cuvettes) to be used, and label microfuge tubes for after electroporation.
- Place 1-2 µL of your plasmid in the bottom of a labelled eppindorf tube. Add 20 µL of just-thawed electrocompetant cells. Mix.
- Open electroporation chambers and pipet 20 µL of bacteria-plasmid mixture, suspended between the bosses (electrodes) of the chamber.
- Handling the chambers carefully, place leaded chamber in a slot in the chamber rack, noting its position. Repeat for each sample. ALWAYS FILL ALL 4 SLOTS WITH CHAMBERS, EVEN IF YOU HAVE LESS THAN 4 SAMPLES TO BE ELECTROPORATED. USE THE DUMMY CHAMBERS LOCATED NEXT TO THE INSTRUMENT OR USE EMPTY CHAMBERS.
- Once 4 chambers are in place, close the safety interlock lid of the chambersafe and secure.
- Plug pulse cable into chamber safe.
- Turn chamber selection knob to your first sample to direct the electrical impulse to the desired chamber.
- With power still OFF, check settings or set to:
- Pulse control = 330 µF
- Low Omega
- FAST charge rate
- For E. coli, set resistance to 4kOmega.
- Turn power switches on for both the power booster and pulse control apparati. Voltage booster will display -16.66.
- Charge pulse control by setting the CHARGE/ARM switch to CHARGE, then press and hold down the UP voltage control button until the voltage reading is ~410 V. Release button, and quickly switch to ARM setting. The voltage will begin to fall slowly. When it reaches 400 V, press TRIGGER button and hold for 1 second. After pulsing, verify that the pulse control unit indicates <20 V. If you hear a cracking noise, your sample probably had too much salt and has exploded all over the inside of the chamber.
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