Frozen Yeast TRAFO Protocol
This Protocol allows You to prepare Frozen Yeast
cells that are competent for transformation after thawing
After Dohmen et al. (1991) Yeast 7: 691-692. See Schiestl et al (1993)
The ability to make yeast cells competent for transformation
in advance allows those laboratories working with few strains
to prepare large batches of cells for later transformation. This
is convenient for laboratories using the two-hybrid and other
similar systems. Yeast cells can be made competent for transformation
by treatment with ethylene glycol and dimethyl sulfoxide (DMSO)
and then frozen in small aliquots and stored at - 70oC.
While the highest transformation efficiencies are obtained with
freshly grown cultures, the moderately efficient transformation
of frozen competent cells saves time. This procedure has been
reported to produce up to 105 transformants / µg plasmid
DNA (Dohmen et al. 1991).
- Grow cells in YPAD (10 ml per transformation) to an OD600
or 0.6 to 1.0. This represents a cell density of approximately
0.6 - 1 x 107 cell/ml.
- Wash the cells in 0.5 vol of 1.0 M. sorbitol, 10 mM Bicine-NaOH
(pH 8.35), 3% ethylene glycol, 5% DMSO (Solution 1), and resuspend
in 0.02 vol of the same solution.
- Freeze the 0.2 ml aliquots slowly using a Nalgene Cryo 1oC
freezing container (Cat. No. 5100-0001) and store at -70oC
- Add 0.1 - 5 µg of plasmid DNA and 50 µg of single
stranded carrier DNA (10 mg/ml) in a maximum volume of 20 µl
on top of the frozen cell suspension.
- Place in a 37oC water bath and mix every 10 -
15 sec until the solution begins to melt. Remove from water bath
and mix until melting is complete.
- Add 1.4 ml of Solution 2 (40% PEG1000 (Roth, Karlsuhe, Germany),
0.2 M Bicine-NaOH (pH 8.35)) and mix by vortexing for 1 min.
- Incubate at 30oC for 1 hr.
- Spin down the cells at 3000 x g for 5 sec and resuspend the
cell pellet in 1.0 ml of Solution 3 (0.15 M NaCl, 10 mM Bicine-NaOH
- Plate an appropriate amount onto SC ommission medium into
a puddle of Solution 3.
Preparation of frozen competent cells from the two-hybrid
strain PJ69-4A using the method above routinely gave results
between 0.9 - 1.0 x 104 transformants/µg.
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