Yeast Trafo /Two-Hybrid System

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Wed Sep 29 14:30:00 EST 1993


------trotta at seqvax.bio.caltech.edu (Chris Trotta) 
writes-------------------------
I am currently trying to get a high efficiency transformation of yeast
with two plasmids.  I have only been able to get as high as 1x10^3
transformants/ug DNA.  This is not high enough to screen a library.

The second part of my post is to get general ideas on the two-hybrid
system.  Stuff like which strains have worked best, methods of 
screening, what hasn't worked etc.  I have begun screening the 
libraries in a His reporter strain YPB2 and have not had any success.
I also used a LexA derived system without any success either.  It 
seems as though there are quite a few false positives in each. In general 
I have recovered 40-50 positive clones which by themselves can activate
transcription.  I am interested in hearing about other screens.

*******************************
Hi Chris!
    I have done a bit of work with the TH system and have found that the 
transformation (TRAFO) efficiency is fairly strain dependent. ]
The best way to squeeze the most transformants out of any particular strain
  is to put in your 
Binding Domain plasmid in first.  I grow this strain on selective media over 
night and then grow for two generations from 5x10^6 cells per ml to 2x10^7 
cells
per ml in YPD or YPAD.  This supercharges the strain for TRAFO but the plasmid
loss seems to be insignificant in most cases.  I then transform in the library
 
and plate onto double selective media.  This seems to work fairly well if you 
use a good TRAFO protocol. May I suggest the following reference;

Gietz, D., A. St Jean, R.A. Woods and R.H. Schiestl. 1992
    Nucl. Acids. Res. 20: 1425.
and the references mentioned within.

The Key to good LiAc/PEG TRAFO is the single stranded carrier DNA!
The preparation is described in :Schiestl and Gietz 1989
                                  Curr. Genet. 16:339-346.
We have recently found that it is no longer necessary to
 phenol extract some carrier DNA preparations (we buy from SIGMA #D-1626) 
The Key to making good carrier DNA is to keep it large!  just sonicate enough
to ensure that you can pipette it after you take a sample and 
boil it and quick cool it in ice water!  Again the key is the bigger the 
better!  I give it (100mls of salmon sperm DNA at 10 mg/ml) 
2 or 3 30 sec blasts of a sonicator with a large sonicator horn at the
 MAX setting for that horn.  Once you have the correct size just freeze in 
aliquots and boil for 10 min before use!  We re-boil occassionally as needed
if the TRAFO freq starts falling.  Also We have found that we get more 
consistant
results with carrier diluted to 1 mg/ml.  

Another very important part of LiAc/PEG TRAFO is the heat shock!
We have shown, that the maximum TRAFO occurs after a 15-20 min heat shock
at 42oC.  If you are doing just the usual 5 min HS you will improve you
efficiency by extending it.  Some people have published that you can get
enhanced trafo by adding ethanol or DMSO to the PEG cell mixture.  But We
find that if we are doing a 20 min HS this rarely adds to levels of TRAFO we 
see.
It may help in specific strains!! so give it a try.  I think it is 
about 10% final concentration of either.  

I find that the strains GGY1::171 and CTY10-5d and Y190 behave differently 
in the LiAC/ssDNA/PEG TRAFO protocol.
GGY1::171 gives about 6 X 10^5 TRAFOs/ug (Fields original strain)
CTY10:5d gives about 9 x 10^5 TRAFOs/ug 
Y190 gives about     5 x 10^6 TRAFOs/ug

I have no experience with the strain YPB2 sorry!

I have used both GGY1::171 and CTY10-5d to pull out many interesting clones 
with the TH system.  There we a number of false positives but there were
also a number of good positives (the percentage of F/G varied with the gene
we usedto screen against). I used the X-gal method of detection!
But am planning to use the HIS selection on my next round! Which should be 
soon
I think the most important thing about false positives is the type of library
you are screening!  If it was made from a GAL4+ host you will get lots of 
GAL4 false positives.  My advice it to get a good library to start with if 
this 
is what you are finding.  If not  then the class of false positives is 
probably
like those described by Stan Fields in an article published in BioTechniques 
14:
pp920-924.  I would fuse a different gene fragment to the DNA binding domain!
I had one case were we found a gene fusion that actually work better when we
truncated it just in from the C terminal end!  May be if you are at your wits
end try a number of different gene fusions to the binding domain.  

Good Luck
Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________




 



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