R. John Lye
rjl6n at virginia.edu
Mon Jul 17 07:33:53 EST 2000
> Hi all,
> we are experienced some problems when preparing e. coli electrocompetent
> cells. May somebody send me a brief protocol for comparing it with the
> mine. I'm specially interested on knowing how many times do you
> concentrate the cells in the final step.
Here's my protocol:
1. Inoculated 2 250 ml flasks containing 50 mls of 2XYT with 0.5 ml of an
overnight culture of JM 109 (I now use JS-5 cells, but the protocol is the
2. Grew culture until OD600 reached 1.16 (this took about 3.5 hours; I had
been aiming for an OD600 of 1.0, but overshot a bit).
3. Chilled flasks for about 1 hour at 4o C (or on ice).
4. Transferred media to 50 ml disposable conical tubes and spun in IEC (or
similar Beckman swinging bucket) refrigerated centrifuge at 3500 rpm (about
2500 X g) for 12 minutes. The centrifuge had been pre-chilled and was run
at 2o C.
5. Poured off supernatant, and resuspended pellets in 1 ml of cold,
autoclaved 10% glycerol. After resuspending the pellets, they were pooled
and then I brought the volume up to 50 mls. with more cold 10% glycerol.
6. Repeat steps 4 and 5 three more times (total of 4 spins), except that
the final volume was 20 mls. After the last spin, I drained the supernatant
fully and aspirated any residual glycerol clinging to the wall of the tube.
Later spins have softer and softer pellets; longer spins might help reduce
7. Resuspended the washed pellet in 3.0 mls of 10% glycerol.
8. Placed 200 microliter aliquots into 0.5 ml microfuge tubes and
quick-froze in liquid N2. The tubes are stored at -70o C.
9. Thawed one tube and used for transformation with supercoiled pUC18 to
check efficiency of this batch. Diluted 1 ng/ul stock 1:100 and used 1 ul
for transformation; plated 50 ul of the 1 ml of SOC (that the cells had been
diluted into after electroporation) and counted colonies after O/N growth on
L + Amp plates. I got 726 colonies, which yields an efficiency of 1.45 X
109/ug supercoiled DNA. Using this protocol, I can routinely get between
0.9 and 4.5 X 109 transformants/ug of supercoiled DNA.
*note: The culture flasks had been autoclaved while filled with Milli-Q
H2O, then emptied and re-autoclaved (or baked at 80o C) dry. This seems to
be important for healthy cells.
Hope that helps,
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