Starter
yeast culture: Inoculate 5-10 ml of liquid medium with a couple of colonies of
the yeast strain and incubate 1-2 days at 225 rpm and 30°C (or 2-3 days at room
temp.) Use SCD –trp +leu
media for AH109 yeast strain containing a bait plasmid; use YPAD for strains
lacking plasmids.
1. In a sterile 250 ml flask, add portion of saturated overnight
culture (usually around 5 ml) to 50 ml of YPAD media to produce an OD600
between 0.10 – 0.20
2. Incubate culture at 30°C and 225
rpm for 3 to 5 hours to obtain an OD600 between 0.4 and 0.7. For
high transformation efficiency cells should go through two divisions and be
harvested in mid-log phase (OD < 0.8).
3. Transfer culture to sterile 50
ml tube and pellet cells using centrifugation at 3000 rpm for 3 min; gently resuspend the cells in 25 ml of sterile water or TE;
re-spin and gently resuspend cell pellet in 0.25 ml
of 0.1M LiOAc/TE.
4. Assemble transformation
reactions in sterile 1.5 ml microfuge tubes (in this
order):
·
0.1 - 0.5 µg
plasmid(s)
·
100 µg boiled carrier-DNA (50 µl
of 2 µg/ul
boiled herring or salmon sperm DNA)
·
100 µl yeast cells
·
Short vortex to mix
·
600 µl LiOAC/TE/PEG (5 ml stock = 0.5 ml 1M LiOAC
+ 0.5 ml 10X TE + 4 ml 50% PEG (MW ~3500))
·
Short vortex to mix
Note: include
a negative transformation control where no plasmid DNA is added.
5. Incubate reactions at 30°C for
30 minutes.
6. Add 70 µl of DMSO to each
reaction. Mix gently by inverting tubes, don’t vortex.
7. Heat shock: Incubate tubes in
a 42°C water bath for 15 minutes.
8. Pellet cells: Spin at 10K rpm
in a microcentrifuge for 20 seconds; pour of
transformation mix and remove remaining liquid with a micropipettor.
9. Resuspend
cell pellet using 0.5 ml of sterile water or YPAD media; stir the pellet by
with a blue micropipette tip to gently resuspend
– don’t vortex.
10. Plate 100 ul
of cell suspension onto SCD (-trp for bait plasmids,
- leu for prey plasmids) selection plates. Spread
cells gently using minimal number of passes with cell spreader. Let plates dry
for a few minutes with lids slightly ajar.
11. Incubate dried plates upside
down at 30°C. Tiny colonies should be seen in 2 days. Pick colonies on day 3 or
4, or wrap plates in parafilm and store in refrigerator.
Notes from Clontech two-hybrid manual:
• Herring
testes carrier DNA (10 mg/ml)
Sonicated, herring testes
carrier DNA in solution can be purchased separately (#K1606), or can be
prepared using a standard method (Sambrook
et al., 1989). Just
prior to use, denature the carrier DNA by placing it in a boiling water bath
for 20 min and immediately cooling it on ice. Use only high-quality carrier DNA; nicked calf thymus DNA is not
recommended.
50% PEG 3350 (Polyethylene
glycol, avg. mol. wt. = 3,350; Sigma #P-3640) prepare with
sterile deionized H2O; if necessary, warm solution to 50°C to help the PEG
go into solution. Autoclave.
10X TE buffer: 0.1 M Tris-HCl, 10 mM EDTA, pH 7.5. Autoclave.
10X LiAc: 1 M lithium acetate (Sigma #L-6883) Adjust to pH 7.5 with dilute acetic
acid and autoclave.
YEAST MEDIA
• YPD medium
20 g/L peptone
10 g/L Yeast
extract
20 g/L Agar (for
plates only)
• For
adenine-supplemented YPD (YPAD), add 15 ml of a 0.2% adenine hemisulfate
solution per liter of
medium (final concentration is 0.003%, in addition to the trace amount of Ade
that is naturally
present in YPD). Or
100 mg adenine powder. Adenine hemisulfate
tolerates autoclaving.
Add H2O
to 950 ml. Adjust the pH to 6.5 if necessary, then autoclave. Allow medium to
cool to ~ 55°C
and then add dextrose
(glucose) to 2% (50 ml of a sterile 40% stock solution).
Note: If you add the
sugar (20 g/l) before autoclaving, autoclave for 15 min; autoclaving for a
longer period of time may cause the sugar solution to darken and will decrease
the performance of the medium.
• SD medium
6.7 g Yeast
nitrogen base without amino acids (or 1.7 g ynb w/o
ammonium sulfate + 5 g ammonium sulfate)
20 g Agar
850 ml H2O
100 ml of the
appropriate sterile 10X Dropout Solution
• Adjust the pH to
5.8 if necessary, and autoclave. Allow medium to cool to ~ 55°C before adding
3-AT, cycloheximide, additional adenine, or X-gal (see below).
• Add the
appropriate sterile carbon source, usually dextrose (glucose) to 2%, unless
specified
otherwise for your
application.
Notes:
• For
3-AT-containing medium, add the appropriate amount of 1 M 3-AT stock solution
after autoclaving and
swirl to mix well. The
concentration of 3-AT used in the medium depends on the specific bait plasmid
used.
• 3-AT is
heat-labile and will be destroyed if added to medium hotter than 55°C.
• 3-AT, a
competitive inhibitor of the yeast HIS3 protein (His3p), is used to inhibit low
levels of His3p expressed in a leaky
manner in some reporter
strains (Fields, 1993; Durfee et al., 1993).