Trivial transformation improvement

ChenHA hzhen at freeuk.com
Wed Feb 23 07:31:39 EST 2000


CSC wrote:

> On 23 Feb 2000, Moez Torki wrote:
>
> > I never do the 37 C recovery step. Indeed bacteria grow slowly (about 20
> > h), but you get the same yield as for the 37 incubation. Try it.
>
> Uuuuhhhh, dangerous !
> This has been on this newsgroup a 100 times. It depends on
> 1. the strain you are using
> 2. what you are trying to clone in which vector
> 3. how many colonies you want: if one will do, o.k. ! But if 0 is too
> little: hmmm
>
> With several strains/experiments I have noticed a three fold rise in
> transformants after a 1-2 hour incubation at 37C before plating.
> Sometimes it was 3 colonies vs. zilch !
> Makes sense too: the little buggers need a little time before they have
> produced enough beta-lactamase. For those of you who don't want to think:
> always include the 37C step.
>

Why don't you do some thinking yourself?  What is the doubling time of
e.coli?  If you incubate it for 2 hours how many cells would you get from a
single transformants?  If you get only 3 colonies, it is likely that you're
only getting a single transformant, what conclusion can you possibly draw with
only a single transformant?  Chance would be a better explanation for your 3
colonies.

Thinking indeed!

>
> Kind regards,
>
> Clemens Suter-Crazzolara, PhD
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