Best Yeast Transformation Protocol (for S. bayanus)
This protocol will allow you to transform a PCR fragment or a plasmid into a yeast
strain.
References:
Oliver Zill (Rine Lab). Modified from:
http://www.umanitoba.ca/faculties/medicine/biochem/gietz/Trafo.html
Protocol:
Day 1:
 Inoculate yeast strains into 5 ml YPD. Incubate overnight at RT/25C.
Day 2:
 Inoculate stationary-phase overnight culture into 50 ml YPD to approx
0.1OD. Shake at RT/25C until OD600 = 0.5-1.0.
o NOTE: This may require another overnight. Alternatively, inoculate
more o/n culture to allow 50mL culture to reach log phase more
quickly. Basically, you just need to start the transformation with a
50mL log-phase culture. I often inoculate a single colony directly
into 50mL YPD, and this will reach log phase by the next morning
(~12-16 hours).
 Harvest the cells by centrifugation at 3000 x g for 5 min.
 Resuspend cells in 1 ml ddH2O. Transfer to a 1.5 ml epi-tube.
 Spin down in microfuge (full speed) for 15sec to pellet cells.
 Discard supernatant and resuspend in ddH2O to a final volume of 1 ml.
 Pipet 100 µl aliquots (one per transformation, plus one per strain for NoDNA control) into 1.5 ml microfuge tubes.
 Spin down in microfuge for 15sec to pellet cells. Remove supernatant.
 Add the following to each yeast pellet:
Reagents
1x
PEG 3500 50% w/v
240 µl
LiAc 1.0 M
36 µl
Boiled SS-carrier DNA
10 µl
(10mg/ml)
PCR product DNA***
50 µl
H2O
24 µl
Total Volume
360 µl
*** Alternatively, 1-3µl of plasmid miniprep, adding more H2O to achieve
360ul total volume.
 Resuspend cells in the mix by vortexing thoroughly.
 Incubate at RT for 10min. Heat shock at 42C for 5 min.
o NOTE: S. bayanus cells appear to be more sensitive to heat shock
than S. cerevisiae. Be sure not to extend the heat shock past 10 min.
 Microfuge at top speed for 15sec. Remove supernatant.


Resuspend cells in 500 µl YPD by pipetting up and down. Be as gentle as
possible with cells at this step.
Plate 250ul onto 2 YPD plates (recovery/pre-growth for antibiotic
selection), CSM/-X (for auxotrophic selection). Grow O/N at RT/25C.
Day 3:
 For antibiotic selection: Replica Plate onto YPD +Antibiotic plates.
o NOTE: standard antibiotic concentrations for S. cerevisiae also work
for S. bayanus. Final conc: Geneticin = 200ug/ml ; Hygromycin =
300ug/ml.
Media/Reagents:
 PEG 3500 50% (w/v)
 LiAc 1.0 M
 Boiled Salmon Sperm DNA (10mg/ml)
 YPD
 Selection Plates