E.coli transformation protocol wanted

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Sat Aug 20 08:13:14 EST 1994


In article <332l7g$cmk at obelix.cica.es>, bb1dopeg at lucano.uco.es (Gabriel
Dorado) wrote:

> Actually, I just saw it in the last NAR issue --have not tried it yet!
> 
> It is based on the novel observation that cultures at high OD (0.94 -
> 0.95) do yield more transformants than when grown to classical OD (0.3
> - 0.4). In short: 5 x10E7 - 1x10E9 vs 2.10E7 - 5x10E8 per microgram
> DNA. Furthermore, you harvest competent bugs at 4 - 8 times higher
> levels.
> 
> Hope it is useful!
> 
> G. Dorado
> bb1dopeg at lucano.uco.es

I tried this yesterday.  And got the results today.  So here are my
results:

  The main problem is that the OD600 grows really fast at the end of the
log phase.  Therefore it is quite a challenge to catch the cells at
0.94-0.95.  I used the TG1 strain which is helthy and grows fast.  I took a
measurement at 0.81 and decided to wait for five minutes until the next
one.  By then the OD was 1.00 and according to the figure in the NAR
article that's too late.  Nevertheless I proceeded and treated the cells
with my usual PIPES/CaCl2/MgCl2/DMSO solution which usually works quite
well.  I transformed 200 ul of the cells with 50 ng of a plasmid of approx.
6 kb. with 90 sec. heat shock, 800 ul SOC, hour at 37 (i.e. just the
standard procedure).  I plated 200 ul and got 26 transformants.  That's not
so swell, about an order of magnitude lower than I usually get when I catch
the cells at 0.4, but still O.K. for most purposes.  The reason is probably
that I cought the cells to late.  So if you want to do this be prepared to
spend some time in front of the spectrophotometer and the incubator. 
Strains that grow slower than TG1 (e.g. XL1-Blue) may also be easier.
  This was my experience and I don't think I'll routinely grow my cells to
0.94.  Still it is good to know that if you are to late to catch them at
the first optimum you can always try the second one.
  I would like to hear if others have had better luck with this than I did.
 If this works well for others I just might try again.


Zophonias



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