Yeast rescue.

Michael Benedik benedik at
Tue Nov 26 14:55:06 EST 1996

In article <57dsdt$1uf at>
jjlapres at (John J. La Pres) writes:

> Here is the scam,  I am currently trying an enhancer trap experiment in which my 
> bait is an enhancer I have isolated from a tissue specific promoter.  I transformed 
> some yeast with this construct and then through a library made from the tissue at 
> this strain.  I then went about rescueing my positive clones.  I have been unable 
> to get any of my transformed bugs to grow.  I use a standard yeast bashing protocol 
> which utilizes 5% triton, Sucrose, Tris and EDTA and glass beads.  After 3 minute 
> vortex, I boil for three minutes, place 100 ul more of lysis buffer (above) and let 
> sit on ice 10 minutes then spin 10 minutes. I remove 100 ul sup and add it to 50 ul 
> of NH4Actetate and let sit on ice 60 minutes.  I follow with 10 minute spin, then 
> ppt DNA from sup usinf EtOH.  I bring this up in 20 H2O and use 1-5 ul for my 
> transformetion into HB101 (my library is Leu marked, 1e9 cfu/ug bugs)  and plate on 
> M9 plates lacking leu and containing AMP.  This is not working.  I know my library 
> is there these yeast grow up in leu - liquid media before the rescue protocol. What 
> am I doing wrong???  Do  HB101's need anything else beside proline, thiamine and 
> the leu my library gives them to grow?  Do they need to be heat shocked longer than 
> a minute? Is there a better rescue protocol out there
> Thanks for any help in advance 
> -- 
> John J. La Pres
> Northwestern University, Evanston, IL.   USA
> jjlapres at

HB101 shouldn't need anything else (I assume you are adding glucose
which is not in your list). Have you tried seeing if you get Amp
resistant transformants on LB + amp lates? Have you also tried some of
your library or vector directly into HB101 to see if it lets it grow on
M9? It is either a strain problem or a transformation problem and you
can easily monitor each individually.

Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at

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