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Transformation Hints

Rob Kirkpatrick kirkpat at ?cc.umanitoba.ca?
Fri May 29 12:46:40 EST 1998


In article <forte-2805980915130001 at 137.53.98.58>, forte at ohsu.EDU (Michael
Forte) wrote:

> Hi,
> 
> I know that this has been discussed here but I am trying to find hints to
> up our transformation efficiency.  Right now, we are hovering around
> 25,000 per ug of DNA.  I have heard reports of over a million per ug but
> even using the now famous TRAFO method, we were not able to improve things
> much, if at all.  I know from experience that transformation efficiency is
> very strain dependent but we would like to at least get 100,000 per ug
> reproducibly, mainly for library screens.  Any hints? Electroporation
> perhap?
> 
> Mike Forte

Hi Mike,

I work in Dr. Gietz's lab at the University of Manitoba and I showed him
your post.  He suggested that you should try some experiments with
variations in heat shock time and carrier DNA concentration to determine
if you can get an optimal condition (there's that bad word -
optimization).  Also, there are a few strains of yeast out there that are
sensitive to lithium and therefore this protocal isn't too great.  Try a
TRAFO where you really limit the exposure to Lithium as much as possible
and see if that helps - for high efficiency, you can't escape the lithium,
but you can limit it's effect.  Let me know if you have any other
questions and feel free to reply to my e-mail if you want.

Hope this helps.

Rob

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