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Need help with Yeast Transformation (ss circles)

M.J. Horsfall mjhf at troi.cc.rochester.edu
Wed Feb 9 11:38:36 EST 1994


I am posting this for someone working in my lab. She is having great
difficulty transforming S. cerevisiae with ssDNA circles. The protocol given
below has worked passably well in the past and we are at a loss as to why it
is not working now. In brief, what we are attempting to do in this procedure
is to transform yeast with a ss vector bearing a single UV photoproduct,
allow the host replicative machinery to copy past the lesion (the vector
contains a 2um ori sequence -although, specifically, only one half of the
normally double-stranded asymmetric ori sequence is present here), rescue
the vector from individual transformants, and transfect E. coli (the vector
is an M13 derivative -i.e., it contains an M13 ori sequence). The problem
does not seem to rest with the DNA/vector. Rather, the yeast cells
themselves or how we make them competent. Even (undamaged) control vectors
are no longer transformable. Also present in the mix are linear vectors
(our efficiency of ligation varies between 40 and 60% normally), an excess
of ss 11mers (used to drop the lesion-bearing sequence into the vector at a
predesigned site), an excess of 51mers (consisting of the complement of
the 11mer flanked by complements of the ends of the uniquely linearized ss
vector), and (added late in the game - post-ligation, pre-transformation) an
excess of anti-51mers used to strip off the scaffold to diminish its use as
a primer for DNA replication - consequently there would seem to be an
abundance of "carrier" DNA present to give a boost to transformation. The
yeast strain we are using is a rad1(del)::LEU2 phr1-1his3/4/5 ura3
maintained on YPD plates. We use the LiCl method (0.1M LiCl in 10mM
Tris-HCl, 1mM EDTA, pH 8.0) with 40% Polyethylene glycol (PEG 3350) in
LiCl/TE (filter sterilized and < 6 months old). Selection is done on 11-10
plates which lack uracil. The protocol is as follows:

Making Competent Yeast

1. Single colonies are picked and grown overnight in 10ml YPD at 30deg C.
2. Samples of the overnight are diluted into 50ml samples of YPD and grown
overnight at 30deg C.
3. At an OD of 50-60 (Klett units, equivalent to 3-5 X 10^7 cells/ml -often
verified via microscope), 40ml of culture is transferred to a 50ml Falcon
tube and spun at 4K for 5min at room temp.
4. The pellet is resuspended in 40ml sterile H2O and spun as above. Note: we
have had somewhat better results when a slight vortex is used to resuspend
the cells).
5. The pellet is resuspended in 8ml LiCl/TE and incubated at 30deg C for
30min with gentle shaking (70 rpm).
6. Cells are spun down at 2K for 5min.
7. The pellet is resuspended in 400ul LiCl/TE gently via a pipette.

DNA Samples

1. Ligated constructs (6ug vector DNA in a 30ul ligation mix) are mixed with
an excess of anti-51mer (6ul at 110 ng/ul).
2. This mix is then dried down (via SpeedVac) to 12ul, to which another 12ul
of sample is added - this gives enough for 4 transformations (each with
1ug).
3. Vector/Scaffold in the Ligation mix are denatured at 85deg C for 3 min.
The tubes are then chilled on ice for 1-2 min (tubes are also centrifuged
briefly to collect condensation).
4. The mix is dispensed as 4 equal aliquots of 3ul (1ug/sample). 

Transformation (under yellow light)

1. 15ul of "competent" ;-) yeast cells are added to each DNA sample and
mixed ever so gently.
2. Tubes are incubated in the dark at 30deg C for 30min without shaking.
3. 180ul of 40% PEG in LiCl/TE is added and mixed in thoroughly by
pipetting. This is an extemely tedious step as PEG is extremely viscous.
4. The cells are "heat shocked" at 42deg C for 5 min.
5. 120ul of H2O are added to each sample and mixed in by pipetting.
6. 100ul of each sample is spread over 11-10 plates (3 plates per sample).
7. After the plates have dried, they are inverted and incubated at 30deg C
for 3-5 days.

As I mentioned above this procedure has worked OK in the past (never
spectacularly, but "good enough"). Does our fatal flaw leap out of the
screen at you? Are there better (tried and true) methods of making yeast
competent? All suggestions are welcome. I should mention that we have played
with electroporation in the past - without much success.

Mike
--
Michael Horsfall Ph.D.             ((           mjhf at troi.cc.rochester.edu
Department of Biophysics            ))          Phone:(716)-275-8703 (Lab)
University of Rochester     5'-GCAAGTCGGAG-3'     Fax:(716)-275-6007(Dept)



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