Transformation of E. coli by Electroporation(electroporation procedure from Cell-PoratorTM Voltage Booster, Life Technologies, Cat. Series 1612)
There are two methods to transform E. coli cells with plasmid DNA - chemical transformation and electroporation. For chemical transformation, cells are grown to mid-log phase, harvested and treated with divalent cations such as CaCl2. Cells treated in such a way are said to be competent. To chemically transform cells, competent cells are mixed with the DNA , on ice, followed by a brief heat shock. Then, cells are incubated with rich medium and allowed to express the antibiotic resistant gene for 30-60 minutes prior to plating. For electroporation, cells are also grown to mid-log phase but are then washed extensively with water to eliminate all salts. Usually, glycerol is added to the water to a final concentration of 10% so that the cells can be stored frozen and saved for future experiments. To electroporate DNA into cells, washed E. coli are mixed with the DNA to be transformed and then pipetted into a plastic cuvette containing electrodes. A short electric pulse, about 2400 volts/cm, is applied to the cells causing smalls holes in the membrane through which the DNA enters. The cells are then incubated with broth as above before plating.
For chemical transformation, there is no need to pre-treat the DNA. For electroporation, the DNA must be free of all salts so the ligations are first precipitated with alcohol before they are used.
Experimental Design :
To determine the efficiency of transformation, a positive control transformation should be done using 1 ng of uncut plasmid DNA, e.g. pUC19. The efficiency of transformation is calculated as the number of transformants/μg of input DNA. A negative control should also be included that contains cells with no added DNA.
A negative control with cells only (no added DNA) should also be included.
For most cloning applications, we use DH5α host cells. These cells are compatible with lacZ blue/white selection procedures, are easily transformed, and good quality plasmid DNA can be recovered from transformants. One notable exception is when transforming with plasmid constructs containing recombinant genes under control of the T7 polymerase. These constructs are typically transformed into DH5α for the cloning phase, but need to be transformed into a different bacterial strain, BL21(DE3) for expression of the recombinant protein (BL21 strains carry the gene for expression of the T7 polymerase).
Electroporation of E. coli:
I. Preparation of E. coli cells for electroporation.
1. Use a fresh colony of DH5α (or other appropriate host strain) to inoculate 5 ml of SOB (without magnesium) medium in a 50 ml sterile conical tube. Grow cells with vigorous aeration overnight at 37°C.
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 37°C until the cells reach an OD550 = 0.8.
3. Harvest cells by centrifugation at 5000 RPM in a GSA rotor for 10 min in sterile centrifuge bottles. (Make sure you use autoclaved bottles!).
4. Wash the cell pellet in 250 ml of ice-cold WB as follows. First, add a small amount of WB to cell pellet; pipet up and down or gently vortex until cells are resuspended. Then fill centrifuge bottle with ice cold WB and gently mix. NOTE- the absolute volume of WB added at this point is not important.
5. Centrifuge the cell suspension at 5,000 RPM for 15 min and carefully pour off the supernatant as soon as the rotor stops. Cells washed in WB do not pellet well. If the supernatant is turbid, increase the centrifugation time.
6. Wash the cell pellet a second time by resuspending in 250 ml of sterile ice-cold WB using the same technique described above. Centrifuge the cell suspension at 5000 RPM for 15 min.
7. Gently pour off the supernatant leaving a small amount of WB in the bottom of the bottle. Resuspend the cell pellet in the WB - no additional WB needs to be added – and the final volume should be about 1 ml. Cells can be used immediately or can be frozen in 0.2 ml aliquots in freezer vials using a dry ice-ethanol bath. Store frozen cells at -70°C.
II. Preparing DNA for Electroporation
DNA for electroporation must have a very low ionic strength and a high resistance. The DNA may be purified by either dilution, precipitation or dialysis.
Purifying DNA by Precipitation:
1. Add 5 to 10 μg of tRNA to a 20 μl ligation reaction in a 1.5 ml tube. Add 22 μl 5M ammonium acetate (or an equal volume of ligation reaction with added tRNA). Mix well.
2. Add 100 μl absolute ethanol (or 2.5 volumes of ligation reaction, tRNA and salt). Ice 15 min.
3. Centrifuge at >12,000 x g for 15 min at 4°C. Carefully decant the supernatant.
4. Wash the pellet with 1 ml of 70% ethanol. Centrifuge at >12,000 x g for 15 min at room temperature. Remove the supernate.
5. Air dry the pellet (speed vac okay but don't overdry).
6. Resuspend the DNA in EB buffer (10 mM Tris-HCl, pH 8.3) or 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. For ligation reactions, it is convenient to resuspend in 10 µl. Use 1 μl per transformation of 20 μl of cell suspension.
1. Mark the required number of micro centrifuge 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 µl of cells to the required number of microfuge tubes on ice. Add 1 µl of the DNA (or ligation reaction) prepared as in Section II.
3. Using a micro pipette, pipette 20 µl 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 kΩ; set the Pulse Control unit to LOW and 330 µF; 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 indicates that 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 30 minutes (for amp), 60 minutes (for Kan) to allow expression of the antibiotic gene. Plate cells on LB medium with appropriate antibiotic or screening reagent (e.g. 100 µg/ml ampicillin, and/or 40 μl of 20 mg/ml X-Gal, XP, and 40 μl of 100 mM IPTG).
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