Electrocompetent Cells

simon day sjd at mole.bio.cam.ac.uk
Fri Feb 9 03:47:51 EST 1996


In article <n1388353683.46505 at msmtp.idde.saci.org>,
brett_beitzel at msmtp.idde.saci.org ("Brett Beitzel") wrote:

> Subject:Electrocompetent Cells                     Date: 2/8/96
> 
> Does anyone know of a E. coli strain that is highly electrocompetent and
> LacI^q?  I need to get transformation efficiencies around 1x10^10 per
> microgram.  With the strain that I am using currently I get around 1x10^8 per
> microgram, but it has an F plasmid, and I have heard that this may reduce the
> electrocompetency.
> Also, anybody have any time-tested protocols for obtaining very highly
> electrocompetent cells?
> 
> Thanks for the info,
> Brett
> brett_beitzel at msmtp.idde.saci.org
> Cancer Therapy and Research Center
> San Antonio, TX

Hi

If you look in the GIBCO-BRL CATALOGUE FOR 96-97 YOU WILL SEE THAT
THEY SELL A STRAIN THAT IS ELECTRO COMPETENT AND IS Lac1 and has efficiency
of >1*10exp10 transformants per ug (Electromax DH12S). I am not sure that
these cells will be of any use,but they have the appropriate genotype and 
effeciency.

The protocol which I use to produce electrocompetent cells uses 10% glycerol

1) Inoculate 200ml of SOB(no Mg) with about 200ul of an overnight culture
of cells

2) Grow at 37 deg with shaking until the O.D. @550nm is about 0.7 ( No
higher than 0.8)

3) Spin down at 5000 rpm for 10 minutes 

4) Remove supernatant and resuspend pellet in a volume equal to the original
culture (i.e. 200ml) with 10% glycerol.

5) Spin @ 5k for 10 minutes

6) repeat 4

7) Spin @ 5k for 10  minutes

8) Carefully pour of the supernatant and resuspend cells in the remaining 
volume of glycerol.Pool the cells in a small centrifuge bottle

9) Spin @ 7k for 10 minutes

10) Pour of the supernatant and resuspend cells in 10% glycerol,using a
volume of 2.0ml per liter of initial culture

11) Aliquot into microfuge tubes and freeze quickly in dry ice/ethanol bath
    Store at -70.


Hope this helps

-- 
SIMON DAY
UNIV OF CAMBRIDGE
DEPT OF GENETICS
CAMBRIDGE



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