Biology Reference
In-Depth Information
A faster but less effi cient protocol to extract protein is as
follows:
1. Grow and recover the cells as described above.
2. Resuspend the pellet in 50
l of 2× Laemmli sample buffer.
The cells can then be broken by freezing on dry ice followed
by boiling for 5 min prior to loading on an SDS polyacryl-
amide gel (about 15
μ
μ
l/lane).
To evaluate the toxicity on yeast growth produced by the
expression of the bait, compare the size of the yeast colonies trans-
formed with pGBKT7 (empty vector) or pGBKT7 + bait. If the
bait is toxic, the colonies containing the bait vector will be signifi -
cantly smaller than colonies containing the empty pGBKT7 vector
(
see
Note 3
).
As a fi rst step for any two-hybrid screen, it is imperative to confi rm
that your bait does not autonomously activate the reporter genes
in the absence of a prey protein.
Testing the Bait
for Autoactivation
1. Transform yeast cells with 300 ng of the bait vector and inocu-
late onto plates of MM without tryptophan to select the trans-
formants (100
l of 10
−1
and 10
−2
dilution).
2. Use sterile toothpicks or inoculating lops to transfer (make
small streaks of 2-3 mm in length of each colony) at least fi ve
yeast colonies onto the following media:
(a) IM-LS, IM-MS and IM-HS (add leucine if cells are trans-
formed only with the bait plasmid) to detect autoactivation.
μ
(b) MM without tryptophan to confi rm the presence of the
plasmid and check yeast growth.
3. Invert the plates and incubate at 30 °C until colonies appear
(3-5 days). Growth should be detected only in MM, otherwise
the bait clone autoactivates (
see
Note 4
).
Most cDNA libraries contain over 10
6
individual cDNAs in
AD-
GAL4
vectors. To isolate the less abundant cDNAs it is
required to obtain 2-3 × 10
6
yeast transformants. Because most
common yeast two-hybrid strains transformation effi ciency is
around 10
5
transformants per
3.1.2 Library Screening
by Co-transformation
g of library plasmid DNA, a large-
volume transformation or several individual transformations are
usually required to obtain the desired number of total transfor-
mants. An exploratory transformation should be performed with
the selected strain to determine the transformation effi ciency.
A yeast transformation protocol readjusted to obtain up to
3 × 10
7
co-transformants is described (Fig.
2
):
μ
1. Streak an YPDA agar plate with cells from a frozen yeast stock.
Incubate the plate upside down at 30 °C until colonies appear
(~3 days).
