M.7: TRANSFORMATION OF E. COLI BY ELECTROPORATION
(Modified from Cell-PoratorTM Voltage Booster Users Manual, Life Technologies, Cat. Series 1612.)
I. Preparation of E. coli cells for electroporation.
- SOB medium
- E. coli host strain such as DH5à
- 5 M ammonium acetate
- 100% ethanol
- 70% ethanol
- 0.5X TE
- SOC medium
- transformation plates
1. Use a fresh colony of DH5à (or other appropriate host strain) to inoculate 50 ml of SOB (without magnesium) medium in a 500 ml flask. Grow cells with vigorous aeration overnight at 37oC. 2. Dilute 2.5 ml of cells into 250 ml of SOB (without magnesium) in a 1 liter flask. Grow for 2 to 3 hours with vigorous aeration at 37oC until the cells reach an OD550 = 0.8. 3. Harvest cells by centrifugation at 5000 RPM (2,600 g) in a GSA rotor for 10 min. (Make sure you use autoclaved bottles!). 4. Wash the cell pellet by resuspending in 250 ml of sterile ice-cold WB (10% redistilled glycerol, 90% distilled water, v/v). Centrifuge the cell suspension at 5,000 RPM for 15 min and carefully pour off the supernate as soon as the rotor stops. Cells washed in WB do not pellet well. If the supernate is turbid, increase the centrifugation time. 5. Wash the cell pellet a second time by resuspending in 250 ml of sterile ice-cold WB. Centrifuge the cell suspension at 5000 RPM for 15 min and pour off the supernate. 6. Resuspend the cell pellet in WB to a final volume of 1 ml. Usually no additional WB needs to be added to the cell pellet; it can be resuspended in the WB that remains in the centrifuge bottle. Cells can be used immediately or can be frozen in 0.2 ml aliquots in microcentrifuge tubes using a dry ice-ethanol bath. Store frozen cells at -70oC.
For best results, DNA for electrotransformation must have a very low ionic strength and a high resistance. The DNA may be purified by either precipitation or dialysis. Purifying DNA by Precipitation: 1. Add 5 to 10 ug of tRNA to a 20 ul ligation reaction. Add 20 ul 5M ammonium acetate. Mix well. 2. Add 100 ul absolute ethanol. Ice 15 min. 3. Centrifuge at>12,000 x g for 15 min at 4oC. Carefully decant the supernatant. 4. Wash the pellet with 60 ul of 70% ethanol. Centrifuge at>12,000 x g for 15 min at room temperature. Remove the supernate. 5. Air dry the pellet. 6. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.
II. Preparing DNA for Electroporation
III. Electroporation. 1. Mark the required number of microcentrifuge tubes. Place the required number of Micro-electroporation Chambers on ice. Fill the temperature control compartment of the Chamber Safe with ~250 ml of ice-water slurry and place the Chamber Rack in the Chamber Safe. 2. Thaw an aliquot of cells that have prepared as in Section I and aliquot 20 ul of cells to the required number of microfuge tubes on ice. Add 1 ul of the DNA (or ligation reaction) prepared as in Section II. 3. Using a micropipette, pipette 20 ul of the cell-DNA mixture between the bosses in a Micro-Electroporation Chamber. Do not leave an air bubble in the droplet of cells; the pressure of a bubble may cause arcing and loss of the sample. Place the chamber in a slot in the Chamber Rack and note its position. Repeat the process if more than one sample is to be pulsed. Up to 4 samples can be placed in the Chamber Rack at one time. Handle the chambers gently to avoid accidentally displacing the sample from between the bosses. 4. Close the lid of the Chamber safe and secure it with the draw latch. 5. Plug the pulse cable into the right side of the Chamber safe. 6. Turn the chamber selection knob on top of the Chamber Safe to direct the electrical pulse to the desired Micro-Electroporation Chamber. 7. Set the resistance on the Voltage Booster to 4 kohms; set the Pulse Control unit to LOW and 330 uF; double check connections. 8. Charge the Pulse Control unit by setting the CHARGE ARM switch on the Pulse Control unit to CHARGE and then pressing the UP voltage control button until the voltage reading is 5 to 10 volts higher than the desired discharge voltage. For E. coli, the standard conditions are 2.4 kv, which means setting the Pulse Control unit to 405 volts (400 volts is the desired discharge voltage + 5). The voltage booster amplifies the volts by ~6-fold such that the total discharge voltage is 2400 volts, or 2.4 kv. The actual peak voltage delivered to the sample will be shown on the Voltage Booster meter after the pulse is delivered. 9. Set the CHARGE/ARM switch to the ARM position. The green light indicatesthat the unit is ready to deliver a DC pulse. Depress the pulse discharge TRIGGER button and hold for 1 second. NOTE: The DC voltage display on the Pulse Control unit should read <10 volts after a pulse has been delivered. If not, discharge the capacitor using the DOWN button. 10. For additional samples, turn the chamber selection knob to the next desired position and repeat steps 8 and 9 until all samples are pulsed.
11. For ampicillin selection, inoculate the samples into 2 ml of SOC medium and shake for 60 minutes to allow expression of the -lactamase gene. Plate cells on LB medium + 50 g/ml ampicillin (and X-gal and IPTG, if appropriate).
SOB Medium: per liter:
- 10mM Tris-HCl, pH 8.0
- 1mM EDTA
2 M Mg++ stock:
- Bacto-tryptone 20 g
- Yeast extract 5 g
- NaCl 0.584 g
- KCl 0.186 g
- Mix components and adjust pH to 7.0 with NaOH and autoclave.
2 M Glucose
- MgCl2 6H2O 20.33 g
- MgSO4 7H2O 24.65 g
- Distilled water to 100 ml. Autoclave or filter sterilize.
For SOB Medium + magnesium:
- Glucose 36.04 g
- Distilled water to 100 ml. Filter sterilize.
For SOC Medium:
- Add 1 ml of 2 M Mg++ stock to 99 ml
- SOB Medium.
- Add 1 ml of 2 M Mg++ stock and 1 ml of 2 M Glucose o 98 ml of SOB Medium.