Electroporation Protocol
Wong Lab
1. Pre-chill cuvettes (0–4 °C) in refrigerator or on ice before using. After chilling, blot off the
excess ice or moisture from the cuvette and holder before inserting into the electroporator.
2. Remove tube(s) of frozen electrocompetent cells from –70°C freezer, and thaw tube on ice.
It is crucial that cells be kept cold during all steps until electroporation is complete.
3. Set up microfuge tubes on ice ahead of time. Divide thawed cells by GENTLY pipetting 50
µL of cells into each prechilled microfuge tube.
Do not add the DNA to the cell solution more than a minute before electroporating. Any
DNases in the cell solution may degrade the DNA of interest and cause low transformation
efficiencies.
DNA may also be directly electroporated from ligation mixtures and enzyme reactions if the
final salt concentration is below 5 mM or its equivalent (typical ligation buffers contain
~50mM salt. If the reaction mixture’s ionic strength is too high, it may be reduced by dilution
(e.g. 1 µL ligation mix per 50 µL competent cells).
Alternatively, ligation mixes can be cleaned up by ethanol precipitation, and the DNA may
be resuspended in PCR-grade water or 10 mM Tris, pH 8. In this way, more of the ligation
products can be added to the transformation reaction.
4. Switch on BioRad 2510 Electroporator. Set the voltage with the aid of the SET VOLTAGE
and arrow keys. It is possible to select the voltage values used most frequently (i.e., 1,800
V and 2,500 V) directly by pressing both arrow keys simultaneously. The voltage first jumps
from 0 to 1,800 V, then from 1,800 to 2,500 V and then back to 0 V. E. coli typically require
1800 V.
5. Quickly transfer cells with added DNA to a chilled cuvette. Ensure no bubbles in cuvette.
6. Insert the cuvette into the cuvette holder of the electroporator. Push the cuvette holder into
the electroporator until it clicks into position.
7. The charging/discharging procedure is started by pressing the PULSE key twice. During
charging (approx. 8 seconds at maximum voltage), "Chg" appears in the display. After
discharge, the unit will beep signaling that electroporation is concluded.
8. Remove the cuvette, and GENTLY add 500 µL LB broth. Transfer cells to sterile microfuge
tube, and shake in 37°C incubator for 1 hour minimum. Quick resuspension helps to
minimize cell death.
9. Immediately wash out cuvettes with three distilled water rinses and three 75% ethanol
rinses. Air-dry in sterile hood (wear clean gloves to handle). Individually-wrap in UVirradiated aluminum foil.
9. Spread 100 µL of cell suspension per plate of selective medium (e.g. LB/S-Gal/Ap).
Incubate plates at 37°C for 24-48 hours.