Quick and Easy TRAFO Protocol
This Protocol allows for TRAFO with any Yeast cell
Referenece; Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION
OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology
All solutions used in this protocol are described
in the TRAFO Solutions Page
When just a few transformants are sufficient, such as the
transformation of a test plasmid or a GAL4BD fusion plasmid,
the following protocol can be used. This procedure is very flexible
and can be applied to yeast cells from a number of different
sources; however, for best results, use cells from a freshly
Rapid Transformation Protocol
1. Inoculate the yeast strain in a 2 cm2 patch onto YPAD agar
(YPD 12 supplemented with 100 mg adenine hemisulphate per liter)
and incubate overnight at 30C. Alternatively, the yeast strain
can be inoculated into 5 ml of liquid medium (2x YPAD or SC selection
medium and incubated on a shaker at 30C and 200 rpm.
1. Heat a tube of carrier DNA in a boiling water bath for 5 min
and then chill in ice/water.
2. Scrape a 50 ml blob of yeast from the YPAD plate and suspend
the cells in 1 ml of sterile water in a 1.5 ml microcentrifuge
tube. The suspension will contain about 5 x 108 cells.
Cells grown overnight in 2x YPAD broth will reach a titer between
1 and 2 x 108/ml; the titer in SC medium will be about
5 x 107/ml. Harvest 2 ml of a YPAD culture and 5 ml
of a SC culture. Note: cells in log phase growth on agar or in
liquid medium will transform with high efficiency.
3. Pellet the cells at top speed in a microcentrifuge for 30
sec and discard the supernatant.
4. *Add the following
components of the Transformation Mix to the cell pellet in the
Be sure to vortex mix the carrier
DNA before pipetting it.
| 1. PEG 3500 50% w/v
| 2. LiAc 1.0 M
| 3. Boiled SS-Carrier
DNA (2 mg/ml)
| 4. Plasmid DNA (0.1
to 1 µg) plus water
| Total Volume
5. Incubate the tube in a water bath at 42°C for 40 to 60
min. Many laboratory strains will yield up to 1 x 105
transformants/mg plasmid after 60 min incubation. Extending the
time at 42°C to 180 min increases the yield to > 1 x 106/µg
with some strains.
6. Microcentrifuge at top speed for 30 sec and remove the Transformation
Mix with a micropipettor.
7. Pipette 1.0 ml of sterile water into the tube and resuspend
the cells by stirring with a micropipette tip and then vortex
8. Pipette 10 and 100 µl samples onto plates of appropriate
SC selection medium, incubate at 30°C for 3-4 days and isolate
transformants. The 10 µl samples should be pipetted into
100 µl puddles of sterile water.
This protocol can be used with cultures that have been stored
at room temperature or in a refrigerator. The yield will be reduced
with older cultures but will generally be sufficient to isolate
a number of transformants of the desired genotype.
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