Biology Reference
In-Depth Information
To transform yeast with the linear DNA fragment containing modified tubulin,
grow a 5-ml yeast culture in YPD media overnight at 30
C with shaking/rotating.
The following day, dilute the near-saturated culture 1:50 into freshmedia and continue
shaking until cells enter log phase; 4 h is usually sufficient with typical growth rates.
Use 10-20 ml of culture per transformation. Harvest cells by centrifuging 5 min at
g
and wash by resuspending in 1/10 original volume of sterile 0.1 M LiAc
in TE buffer. Harvest cells again and resuspend in 1/100 original volume of 0.1 M
LiAc inTE. For each transformation, combine in a sterile 15-ml tube the purified linear
DNA fragment containing the mutated tubulin gene (
1000
0.5
m
gin
30
m
l) with 10
m
l
<
single-stranded salmon sperm carrier DNA. Subsequently, add 100
l LiAc-treated
yeast cells and mix gently. Add 0.7 ml sterile 40% PEG, 0.1 M LiAc in TE and
mix well. Incubate for 30 min at 30
C with shaking. Heat-shock the cells for
20 min in a 42
C waterbath. Add 10 ml YPD and allow cells to recover for 2 h at
30
Cwith shaking. Harvest cells, resuspend in
m
0.5 ml YPD, spread onto appropriate
selection plates, and incubate at least 2 days at 30
C to allow colony growth.
When colonies reach sufficient size, potential transformantsmust be taken through
two rounds of single-colony isolation on appropriate selection media to generate
clonal strains. To isolate single colonies, streak cells across a fresh plate with a sterile
loop or wooden toothpick to obtain sparse colony growth. For long-term storage, iso-
lated clones should be grown in YPD or appropriate selection media, resuspended in
sterile 15% glycerol, and stored at
80
C in cryovials. To revive, transfer a small
scraping from the still frozen tube onto a fresh plate using a sterile wooden applicator.
Note
: After several freeze-thaw cycles, incubate salmon sperm DNA aliquots at
95
C for 10 min and cool on ice to enrich single-stranded fragments.
22.2.3
Verification of tubulin mutations in yeast
Although fragment-mediated gene replacement is efficient, it is possible for the pro-
totrophic marker to integrate into the genome independently from the tubulin ORF.
Thus, it is essential to verify that the isolated clonal strain carries the desired muta-
tion before proceeding with analyses. Verification can be accomplished by PCR am-
plification and sequencing of the tubulin locus. Primers should anneal
50 bases
outside the tubulin ORF. Genomic template DNA is readily obtained using commer-
cial products, for example, the Yeast DNA Extraction Kit (Pierce). Colony-based
PCR can be a useful alternative when dealing with many samples, although it is less
robust than PCR from purified genomic DNA. Using a sterile pipette tip, resuspend a
small portion of a fresh colony in 10
l of 20 mM sterile NaOH in a PCR tube.
Incubate in a thermocycler for 10 min at 95
C. Use 3
m
m
l of the NaOH-treated cells
m
as a template in 25-
l reactions and perform PCR using standard conditions.
Sequence the PCR product to verify presence of the mutation. If the mutation is
in a diploid strain, the sequencing chromatogram should reflect the presence of both
the wild-type and mutated alleles as a double peak at the position corresponding to
the mutation (
Fig. 22.2
).
