Biology Reference
In-Depth Information
12. Plate the remainder of the transformation mixes onto
24 cm × 24 cm IM-LS plates to select for the induction of
HIS3. There is no need to spin the cells or remove the PEG.
To achieve an even distribution of cells, pour about 100 sterile
glass beads (4 mm diameter) onto the plate with the cells.
Gently roll the beads around the plate to distribute the trans-
formation mix. This technique works best when the surface of
the plates is not too wet so that the medium absorbs the trans-
formation mix. To achieve this moisture content, put newly
solidifi ed plates into a laminar fl ow hood with the lids ajar for
about 1 h before plating.
13. Incubate the plates at 30 °C. Continue incubation until colo-
nies are 1-2 mm in diameter, which should take a total of
approximately 3-5 days.
14. Use sterile toothpicks or inoculating lops to transfer all of the
grown colonies onto 10 cm diameter IM-LS plates to confi rm the
result and isolate the colonies. Make small streaks of 2-3 mm in
length of each colony. Incubate the plates at 30 °C for 2-4 days.
If the colonies are closely spaced it will be necessary to streak to
single colonies to separate the different co-transformants.
15. Collect the yeast from one streak using a sterile toothpick and
resuspend them in 100
l of deionized sterile water in 1.5 ml
microcentrifuge tubes. Add 5
μ
μ
l of each suspension to plates
with the following media:
(a) IM-HS to select for the induction of
ADE2
and
HIS3
.
(b) IM-MS to select for the induction of ADE2.
(c) IM-LS to select for the induction of HIS3.
(d) CM to check cell growth.
Incubate the plates at 30 °C for 2-7 days. Positive interactions
will be determined by comparison with the growth of the positive
and negative yeast two-hybrid controls on each media (
see
Note 4
).
1. To rescue the library plasmid, inoculate 10 ml of CM media
with the positive yeast colony and incubate at 30 °C overnight.
Centrifuge the culture at 3,000 ×
g
for 4 min. Remove all super-
natant and resuspend the pellet in 200
Rescue of the Prey Vector
l of DNA breakage
buffer. Alternatively use sterile toothpicks or inoculating lops to
transfer to make small streaks of 2-3 mm in length in a plate of
CM media. Incubate at 30 °C overnight and resuspend a tooth-
pick worth of cells in 200
μ
μ
l of DNA breakage buffer.
2. Add a scoopful of glass beads (425-600
μ
m of diameter) and
l of phenol-chloroform.
3. Vortex for 4 min.
4. Centrifuge at 13,000 ×
g
during 5 min at room temperature.
Transfer the upper aqueous layer to 1 ml of cold ethanol
200
μ
