HELP ... yeast transformations


Sat Jul 30 16:04:03 EST 1994


In article <3102df$4d2 at yama.mcc.ac.uk>, Julie <JANDREWS at fs1.pa.man.ac.uk>
wrote:
> 
> We are currently using the Lithium acetate method to transform an
> E.coli shuttle plasmid into S.cerevisiae. However, contrary to what
> we would expect, we find that we do not see any improvement in the
> transformation efficiency when we include a heat shock of 42 degrees
> for 15 minutes. We are finding that with or without the heat shock we
> routinely get about 200 transformants from 1ug of DNA added to 40ul
> of competent cells. WE EXPECTED TO SEE ABOUT A TENFOLD IMPROVEMENT WITH
> THE HEAT SHOCK STEP INCLUDED.
> 

Julie,
	I have found that transorfmations using the LiOAc method can be highly
strain and plasmid dependent.  I recently optimized the protocol for my
strain/plasmid and found that the following procedures increased
transformation efficiency:
- add DMSO to a final concentration of 10% before heat shock
- try different heat shock durations (my strain starts to die after 10
minutes)
- check length of incubation in PEG (my strain transforms best after 15
min, but some strains require prolonged incubation)
- use a final concentration of 33.3% PEG
- include 0.1% glucose (or your preferred carbon source) in all buffers
- plate cells in 1M sorbitol, 0.1% glucose
- don't concentrate cells more than 100 fold (ie resuspend cells from a 100
ml culture in 1 ml)
While some of the improvements seen with these additions may be specific to
my strain, I hope this will be helpful.  I too found that heat shocking
without DMSO produced only a small effect.
Yours,
	Jason Brickner



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