Posted on June 2nd, 2016 by UNM CC
8/17/2011 Shyamala Mohan
Protocol adapted from Molecular cloning 3rd edition, Joseph Sambrook and David W. Russell
Electrocompetent cells are prepared by growing cultures to mid log phase, washing the bacteria at low temperature and resuspending them in a solution of low ionic strength containing glycerol. The starting material can be a vial of compentent cells from a prior preparation. If there is any uncertainty about the purity of the cells they should first be cultured by streaking onto LB and LB + Amp plates. Untransformed competent cells should only grow on LB plates. Single colonies can then be picked and grown in LB media to generate a culture. The final product of this preparation are aliquots of bacteria that can then be be used for DNA transformation using electroporation.
DH5α E. coli, or comparable strain
LB culture medium
Sterile 10% glycerol in dionized water (40 mL of glycerol in 360 mL water)
Ice water bath
LB agar plates
LB + Amp agar plates
Sterile centrifuge bottles (250 or 500 mL)
Spectrometer and cuvettes
Dry ice/ethanol bath
- Thaw 50 µl of frozen DH5α competent cells and inoculate it in 500 mL of LB media. Inoculate the flask at 37ºC with agitation. Measure the absorbance (A600) of the growing bacterial culture every 20 minutes. To estimate the finish time, wait until the cultures reach exponential growth and then graph the A600 on semi-log plot (time vs log A600) to estimate the time for an A600 of 0.4.
- In preparation for the next step, place the centrifuge bottles in ice-water bath. Place the 400 mL of 10% glycerol and 500 mL water in ice bath. Turn on the Sorvall centrifuge and cool the chamber to 4ºC with the rotor in place. Also precool a table-top centrifuge. These steps are important to keep the bacteria cold.
- Bacteria are most competent in mid log-phase (A600 = 0.4-0.5). Measure the A600 by removing 0.5 mL into a plastic cuvette. Record the absorbance at 600 nm. When the A600 > 0.4, transfer the flasks to an ice water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly.
- Transfer the cultures to ice cold centrifuge bottles. Harvest the cells by centrifugation at 1000 g (~2500 rpm) for 15 minutes at 4ºC. Decant the supernatant and resuspend the cell pellet in 500 mL of ice cold pure H2O.
- Repeat the spin and wash of the bacteria, but this time resuspend the bacteria pellet in 250 mL of ice cold 10% glycerol.
- Repeat the spin and wash of the bacteria, but this time resuspend the bacteria pellet in 10 mL of ice cold 10% glycerol. Meanwhile place 15 mL conical tubes on ice.
- Harvest cells by centrifugation at 1000 g for 20 minutes at 4ºC in a table top centrifuge. Carefully decant the supernatant and resuspend the pellet in 1 mL of 10% ice cold glycerol. Meanwhile, place 1.5 mL Eppendorf tubes on ice.
- For storage, dispense 50 µL aliquots of the cell suspension into sterile ice-cold 1.5 mL microcentrifuge tubes, drop into a bath of dry ice with ethanol (to freeze the competent cells immediately) and transfer to a -80ºC freezer. Record the location of the cells in the MPD.
- On the following day test the competency of the cells by transforming aliquots with successive dilutions of pBS plasmid vector. Count the number of colonies on a plate and calculate the number of colonies per µg of DNA transformed. Highly competent cells should yield 108 to 109 colonies per µg of DNA transformed. High levels of competency are needed for difficult cloning procedures, but lower competency cells can still be used for maintaining plasmid stocks.