frozen competent yeast

Scott Vande Pol sbv at pop.cwru.edu
Mon Jan 22 12:48:02 EST 1996


Thanks to all of you who E-mailed me last week of this topic.  I tried
several recipies and the clear winner for routine transfections was a one
step transformation protocol recommended to me by Christian Fritz 
(cfritze at cts.com) (Christian Fritze).  Thank you Christian!  Here is the
protocol for those of you who also might like it:


One Step Yeast Transformation of yeast in Stationary Phase
Chen, D. C.,Yang, B. C., Kuo, T. T.
Curr Genetics 21: 83-84.  1992

Transformation Buffer:

Litium Acetate,  0.2N
PEG 3350         40%
DTT                  0.1M
Denatured DNA  500ug/ml

Grow yeast overnight in YPD media.  If grown in selective media, in AM grow
for several hrs in YPD.  Can now store at 4ûC for several days or freeze
aliquots of yeast in 15% glycerol at -20 or -70.  An overnight grown in YPD
is about 3 OD/ml or 2X108 /ml. Want 5X107 cells per transfection----about
0.25 mls.

1. Thaw the transformation buffer on ice.

2.  Spin down 0.25 ml of stationary phase yeast in microfuge tube (3-4
seconds).  Remove the supernatant, add the plasmid DNA to be transfected (1
ug) and 0.1 ml of transformation buffer.  Resuspend the yeast in this
mixture and incubate in a heat block at 45 C for 30-60 minutes.  Mini-prep
DNAs must be etoh ppted prior to transfection as the Triton X100 in our
minis is toxic to yeast.

3.  Plate directly onto selective media, incubate at 30 C for 2-3 days.

Expect 103 to 105 colonies / ug plasmid DNA.  Log growth phase cells give
similar efficiency, frozen and or stored cells about a log lower effiency.



-- 
Scott Vande Pol
Case Western Reserve University
Department of Pathology
Cleveland Oh. 44106
sbv at pop.cwru.edu



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