Trivial transformation improvement

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed Feb 23 10:34:42 EST 2000


Rafael Maldonado wrote:

> ChenHA wrote:
> >
> > 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.
> >
>
> Not everything in Science is thinking. There is also knowledge.
>
> Bacteria don't grow well at high cell concentration, as in the recovery
> step after trasformation. Doubling times in a growing culture cannot be
> extrapolated to cells recovering from a transformation step,
> concentrated 10 to 50 fold of a overnight culture, and after a sort of
> stress condition.
>
> So, one hour recovery incubation does not give you siblings after
> transformation.
>

Some people think it is bad when cells double after transformation, but
why should
it? Of course, if you are constructing a library, it is desirable to
have no
doublings before plating. In all other cases, it might even be
beneficial to have
some doublings (even though I agree with Rafa that it is unlikely in the recovery
step). That´s because usually only a portion of the transformation is
plated (say
50ul of 1ml), so you´re likely to miss clones if they are unique.  I
actually never
care about the exact time of recovery (somewhere between 1h and
"uuhhhh....I forgot
my tranformation"), and never had any problems with it.

Frank




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