Transformation of E. coli
This procedure prepares glycerol stock cultures of bacteria
for electroporative transformation.
Preparation of Electrocompetent Cells
- 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)
- Add colony of desired bacterium to 25 mL LB flask; grow
- 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
- 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
- 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
- Life-Technologies Cell Porator
- 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.
- 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
- Open electroporation chambers and pipet 20 µL of
bacteria-plasmid mixture, suspended between the bosses (electrodes) of
- 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.
- For additional samples, move chamber select knob and
repeat electroporation as above.
- Turn off power buttons on both the power booster and
pulse contol and disconnect cable on chamber safe. Remove suspended cells
from chambers with a pipet and place in 1 mL rich media (SOC) to recover
for 1 hr. at 37 °C in a shaker.
- Plate 150 µL on appropriate plates (with antibiotic
that corresponds to the resistance offered by your plasmid). Let grow overnight
in 37 °C incubator.
- You now can screen colonies for the plasmid.
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