Yeast rescue.

[Michael Benedik]

In article <57dsdt$1uf at news.acns.nwu.edu>
jjlapres at merle.acns.nwu.edu (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 merle.acns.nwu.edu
> 

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 uh.edu