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LiOAc YEAST TRANSFORMATIONa
(see notes at end)
Use STERILE TECHNIQUE throughout.
1. Inoculate a 10 ml overnight culture with a
single yeast colony. If starting with a yeast strain with no
plasmids, use YPD. If starting with a strain that already has one or
more plasmids use liquid drop-out media lacking the appropriate
nutrient.b Incubate at 30oC in roller drum at
top speed overnight. b
2. Measure the OD600 of the
overnight culture.c Make an appropriate dilution of the
overnight culture into 50 ml of media in a sterile 250 ml flask so
that the final OD600 is 0.2.
3. Incubate at 30oC, shaking at
200 rpm, until OD600 = 1.0
4. Transfer culture (using sterile
technique!) to a sterile 50 ml polypropylene tube (Oakridge tube).
Spin in clinical centrifuge at 3000 rpm, 2 min. This and all
following manipulations are done at room temperature.
5. Dump off media (quickly and carefully) and
add 10 ml sterile water. Resuspend cells by swirling or gentle
vortexing.
6. Pellet cells in clinical centrifuge at
3000 rpm, 2 min. Pour off water.
7. Add 5 ml of LiOAc/TE solution. Resuspend
cells by swirling or gentle vortexing.
8. Pellet cells in clinical centrifuge at
3000 rpm, 2 min. Pour off LiOAc/TE.
9. Add 0.25 ml of LiOAc/TE solution.
Resuspend cells by swirling or gentle vortexing.
10. For each transformation aliquot 50
µl of cells to a sterile eppendorf tube. ALWAYS set up a control
tube that will get no DNA.
11. Add plasmid DNAd. [For
higher efficiency (e.g. for library
transformations
e) add DMSO to 10% (e.g. ~ 6
µl)]. Mix gently. Add 300 µl 40% PEG in LiOAc/TE. Mix
by inverting two or three times.
12. Do not vortex. In the presence of PEG
yeast cells are more fragile.
13. Incubate at 30oC for 30 min.
Take this time to check the temperature of the 42oC, and
warm and label plates. Remove cells to room temperature.
14. Incubate at 42oC for exactly
15 min. Remove to room temperature.
15. Plate 200-300 µl, including the bulk
of the cells, onto appropriate plates. Incubate at 30oC
with plates right side up. Turn plates over after 1 day.
NOTES
a This method is based on
the procedure of Geitz et al., 1992 Nucleic Acids Research
20:1425.
b When using YPD, 12 to 16 hours
is sufficient for a 10 ml overnight culture. Growth in drop-out media
may take much longer so the "overnight" culture may be started in the
morning for the following day. Alternatively, a 50 ml overnight
culture may be started - which may or may not need to be diluted the
next day.
c Spectrophotometer measurements
are only linear at OD600 between ~0.05 and 1.0. If the
OD600 of the culture is over 1.0 it should be diluted
10-fold to take the measurement. Note: Ideally one should try
transforming yeast at different OD600 from 0.7 to 1.5 to
see which is best. Spects can differ, and this approach is easier
than counting cells
d Use no more than 1 µg
plasmid DNA/50 µl of cells. For routine transformations, 5-10
µl of miniprep DNA or 0.1-0.5 µg maxiprep DNA is
sufficient. For library transformations use 1 µg plasmid PLUS 30
µg carrier DNA (such as Boehringer MB grade fish sperm DNA Cat#
1467140; see also Schiestl and Gietz (1989) Current Genetics
16:339-346).
e
To perform 10 or more transfromations, scale up by using a 200 ml
culture at step 2, and resuspend in 1 ml LiOAc/TE solution at step
9.
SOLUTIONS
LiOAc/TE
For 50 ml, in a sterile 50 ml Falcon
tube.
44.4 ml sterile water
5.0 ml sterile 1 M LiOAc, pH
7.0-7.4
0.5 ml sterile 1 M Tris HCl, pH
7.5
0.1 ml sterile 500 mM EDTA
40% PEG in LiOAc/TE
For 50 ml, in a sterile 50 ml Falcon
tube.
40 ml sterile 50% PEG 4000
5.0 ml sterile 1 M LiOAc, pH
7.0-7.4
0.5 ml sterile 1 M Tris HCl, pH
7.5
0.1 ml sterile 500 mM EDTA
4.4 ml sterile water.
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