Yeast Transformation!!!!!!!!!!!

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Hello to all you Buddies striving to get the most out of your
transformations!!!!

		There have been a number of articles posted to this board discussing
transformation efficiency.  How do I get more etc. What is the best method?
Let me start by saying that all strains are not created equal when it comes
to transformation efficiency when using the different methods!  I have
found that a strain that transforms with a medium efficiency with
LiAc/ssDNA/PEG transforms better with electroporation.  Again If I take all
the strains I normally use in my lab and compare there is a wide difference
in the TRAFO efficiency we see!  There are a few things one can do to
squeese the most TRAFOs out of a strain with the LiAc/ssDNA/PEG method.

	1.	Make good single stranded salmon sperm Carrier DNA!  The key is to keep
the DNA as large as possible (MW) but still be able to handle it after
boiling and quick cooling! As most know that if you dissolve large
molecular weight DNA in TE at 10 mg/ml and boil and quick cool you get a
very stiff gel.  Not good for adding to a TRAFO reaction.  Therefore I make
100mls of 10 mg/ml and once insolution I sonicate for 30 sec with a large
horn  and then test 1 ml of it for the 5 min boil quick cool test.  Usually
it takes 2 -3 30 sec blasts to get the DNA small enough that it will stay a
liquid after the boiling and quick cooling.  I have recently found that
some batches of salmon spern DNA from Sigma donot need to be phenol
extracted to give very good transformation.  If you have having problems
with the 10mg/ml solution make one at 1 mg/ml.  This solution is easier to
handle but increases the volume of the cDNA added to the TRAFO reaction.  I
find that good carrier DNA is very important for good TRAFO efficiencies.  
Fatima mentioned that dirty DNA (DNA with RNA from the plasmid prep) is
better the clean stuff.  Well She is right.  In our first paper Robert
Schiestl and I showed that RNA can also be used as a very effective
carrier.  So dont go to great lengths to clean up your DNA! (Which the
mammalian transfections require)  RNA contamination actually helps.

2.	To get the best TRAFO efficiencies out of your strain one should
optimize a number of variables.  The heat shock is very important.  Most
people dont like to give a 15 or 20 min heat shock at 42¡ but believe me
most strains need it.  If your strain is heat sensitive, reduce the
temperature to 40¡ or 37¡ but do the experiment to see what duration gives
you the best transformation.  You can add DMSO or Ethanol as some
references have eluded to but we have found that if your heat shock is
optimal you get no enhancement by adding these things in the strains we
have tested.  Try it though, it may work for you!

3. Optimize the amount of carrier DNA for your strain.  Some carrier preps
differ and we have have seen some strains that need 75 ug instead of 50 for
the best TRAFO.

4.	Growth conditions are important.  Make sure that your culture is
actively dividing!  Most strains transform best with LiAc after they have
gone through 2 division.  We subculture to 5 x 10^6 cells/ml and let grow
to 2 x 10^7 cells per  ml.  This takes 3 - 4 hrs and is well worth the wait
if you are trying to eek out all the TRAFOs you can.  

5. The % PEG in the TRAFO reaction is fairly important as the peak is quite
narrow.  If it gets too high TRAFOs go down dramatically!  We had a two
month period in my lab were we lost the ability to get good TRAFOs.  We
tracked it down to PEG which was stored with a loose lid and was at a
concentration higher than 50% thus reducing the TRAFO efficiency.  Follow
the TRAFO recipe exactly and make sure your PEG is 50% weight by volume!!! 
Thats 50 gms of PEG 3350 MW in a final volume of 100mls.

6.  If you need to put a library into a strain along with another
plasmid like in the two hybrid system of Fields and Song then it is best to
transform in the bait plasmid first (pMA424 or pAS or equivalent).  Then
grow a 10 ml culture of this strain selectively, to keep the bait plasmid
in the cells, in SC-HIS or TRP to give about 1-2 x10^7 cells/ ml.  I then
dump this 10 mls into 40mls of warm YPAD and grow for 2 generations.  There
is usually negligable plasmid loss in two generations.  Transform as usual
and plate onto double omission medium.  Contrary to some opinion we have
found that transformation of two plasmids from a mixture is only 30 -40%
that of a single plasmid.

7.	Some strains never give good TRAFO by LiAc/ssDNA/PEG.  With these I
would go to another method as suggested by others.  The Electroporation
method of Manivasakam and Schiestl,1993 NAR 21:4414-4415 is a good one. 

8.	Another thing I have found that make a difference is the pH of the
selective medium you are plating on.  We routinely adjust to pH 6.5.  If it
is too acidic this appears to decrease transformation efficiency.  Another
important point that some forget is to keep your SC-minus medium away from
light.  We have traced poor TRAFOs and just plating efficiency to plates
that have been expose to 24 hrs of fluorescent light.  Cover those plate
with a box when drying and keep the cold room light off (if thats where you
store them) or else store them in covered container.  YPAD plates dont seem
to be affected but SC-minus medium is seriously affected.

REFERENCES for all this stuff are

Schiestl and Gietz 1989 Curr Genet 16:339-346
Gietz and Schiestl	1991 Yeast 7: 253-263
Gietz et al 1992 NAR 20 1425
Schiestl et al 1993 Methods:A companion to methods in Enzymology 5:79-85
There are also another other book chapter coming out discussing
 how to TRAFO to the MAX
	Watch for: Molecular Genetics of Yeast (Oxford University Press) Due soon
Also another is planned from CSHLs.

Anyone that has specific questions about TRAFO please feel free to drop me
a message to the address below!

GOOD TRAFOs!!!!!!!!
					
Dan Gietz

**************************************************************************
R. Daniel Gietz Ph.D.
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm250
Winnipeg, Manitoba, Canada R3E0W3
Tel(204)789-3458  Fax 204-786-8712  Email:GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
*****************************************************************************

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