Making Competent Bacterial Cells for Transformation

Leave a comment

DMSO competent




PEG/DMSO transformation (which I’m a fan of, heheh), it’s very easy. The protocol is based on Chung and Miller, 1988.

To prepare competent cells, just grow E. Coli to OD ~0.5, pellet them by gentle centrifugation at 4°C, resuspend in 1/10th of the original culture volume of ice-cold, sterile-filtered TSB buffer (LB pH 6.1 + 10% PEG-3350 + 10 mM MgCl2 + 10 mM MgSO4 + 5% DMSO) and incubate for 10 minutes on ice. Store in 250 or 350 ul aliquots at -80°C.

My lab’s protocol is slightly different from Chung and Miller’s in the transformation part. We add in a chilled eppendorf 20 ul of 5xKCM buffer (0.5M KCl, 0.15M CaCl2, 0.25M MgCl2), the ligation or plasmid (up to 15 ul) and water to 100 ul. Then we add 100 ul of competent cells, leave on ice for 20 minutes, and then at 37°C for half an hour, and plate. No heat shock is needed in this protocol.

I routinely get efficiencies of about 10^7 on DH5a (with supercoiled plasmids). You can refreeze and reuse the cells once, but of course the efficiency lowers a bit.

Good luck,



Second protocol:

The bacterial transformation mix contains:
10% Polyethylene Glycol(PEG) 3350
PEG 3350 is thought to play several different roles in transformation, though nobody really knows for certain. Since both DNA and cell walls are negatively charged, they reject each other. PEG 3350 is thought to function by shielding the charge of the DNA, thereby making it easier to permeate the cell wall. PEG 3350 is also thought to help transport the DNA into the cell, as well as make the cell membrane itself more porous.

5% Dimethyl Sulfoxide (DMSO)
DMSO is sometimes used to treat ailments in humans. In a transformation it is thought to
permeabilize the cell wall. Also, sometimes DNA folds into complex structures that make it more difficult to pass through the cell wall. DMSO also might help to break these structures down.

25mM Calcium Chloride(CaCl2)
Similarly to PEG 3350, CaCl2 is thought to shield and neutralize the negative charge of DNA, thereby making it more likely to enter into the cell.
1. If you received a plate or stab of E. coli HME63, simply use an inoculating loop to
gently scrape out the bacteria and spread it onto a new LB Agar plate. Let the plate
grow overnight ~12-18 hours, or until you see white-ish bacteria begin to grow. Make
sure you are using the LB agar plate, NOT the LB Strep/Kan agar plate. See the
following link for a walk-through of how to streak out bacteria:




Pushing the Limits of DNA Assembly

Leave a comment