Trivial transformation improvement

a.krimer at biosidus.com.ar a.krimer at biosidus.com.ar
Thu Feb 24 07:52:51 EST 2000


I'm sorry Rafa bit I agree with Chen. In fact,  I have performed a recovery time
curve several years ago.
Depending on stress conditions my bacteria needed about 5-15 minutes to recover.
After 15 minutes I have got more clones each time, and after 50-60 minutes,
every 20-25 min (doubling time) I have got approximately twice colonies each
time.
I think it is not so much work to do this work to optimize the recovery time.
Hope this helps.

Alejandro Krimer
R & D Department
Bio Sidus S.A.




Rafael Maldonado wrote:

> 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.

Pardon my language, but this is bollocks!  The cells can grow well at high cell
concentration, everything depends on nutrients and growth conditions.  IIRC, it
is
in fact the exact opposite, cells don't grow well at very low cell
concentration.
If you start a cell culture with very low amount of cells, the lag phase will be
much longer than would be expected with the cell doubling time longer than at
log
phase.  Coli likes the company of other coli, OK?  If you grow coli in a good
fermenter, you can get OD of ~100.  If you look into your plates, what do you
think
you are seeing in the colonies? Discrete individual cells well separated from
each
other?  No, it's coli squashing their bums against each other, piling high
thousands
deep, fimbrae all akimbo.

Note that the genius who said it originally used incubation of 1-2 hours, I
would be
very much surprised if there isn't at least a doubling of cells.

> 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,

Oh well, let's see.  Standard transformation protocol, cell harvested at OD
~0.6,
cell finally concentrated to ~1/10-1/25 (protocols varies, I used 1/12) of
original
cell culture volume in transformation buffer, therefore let's say prepared
competent
cells at density of OD of 10.  Standard transformation procedure: 50 ul of cell
transformed, add SOB to 1 ml, therefore final cell OD ~ 0.5.  What's that you
are
saying about 10 -50 fold concentration of overnight culture?

> and after a sort of
> stress condition.

> So, one hour recovery incubation does not give you siblings after
transformation.

Excuse me while I put my appreciation of your superior knowledge on hold for the
time being.  Until you produce some references that is.  Here's a reference for
you
in the mean time with a quote from the abstract:

Huff et al, (90) Biotechniques 9(5), 570-2, 574, 576-7

--quote--

    ... and an unchanged or even increased transformation efficiency when the
preplating incubation step was omitted.

--unquote--

>

> IMHO...
>
> -Rafa
>
> --
> Rafael Maldonado
> Divison of Genetics
> University of Alicante (Spain)





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