Biology Reference
In-Depth Information
Clontech protocols (Protocol No. PT4084-1, Version No.
PR033493). Protocols for the LexA-based system are available in
http://www.protocol-online.org/
.
The screening process consists of the following steps:
1. Clone and test bait for expression, toxicity and autoactivation.
2. Screen the cDNA library.
3. Confi rm and interpret results.
The bait clone to express
GAL4
DNA-BD fusioned to your gene
of interest can be obtained by cloning it in frame with the
GAL4
DNA binding domain of the bait plasmid pGBKT7 (
see
Note 1
).
Before carrying out the library screening it is strongly recom-
mended to determine whether or not your bait is expressed in
yeast, does not have toxic effects and does not autonomously acti-
vate the reporter genes in the presence of any given prey protein
(autoactivation).
3.1.1 Preparing the
Bait Vector
1. Streak an YPDA plate with cells from a frozen yeast stock.
Incubate the plate upside down at 30 °C until colonies appear
(~3 days). Yeast strains can be stored for up to 2 months at
4 °C on YPDA medium in petri dishes sealed with parafi lm.
However, fresh colonies will give better results when inoculat-
ing a liquid culture.
2. Inoculate one colony into 15 ml YPDA medium in a sterile
50 ml fl ask or tube. Incubate at 30 °C with shaking at 250 rpm
over-night.
3. Transfer an appropriate volume of the over-night culture to
50 ml of YPDA in a 250 ml fl ask to reach OD
600
of 0.2.
Incubate shaking at 250 rpm until the OD
600
reaches 0.8-1
(6-8 h).
4. Centrifuge the cells at 3,000 ×
g
for 4 min at room tempera-
ture. Discard the supernatant and resuspend the pellet in 25 ml
of sterile deionized water.
5. Centrifuge the cells at 3,000 ×
g
for 4 min at room tempera-
ture. Discard the supernatant and resuspend the pellet in 25 ml
of 100 mM lithium acetate.
6. Centrifuge the cells at 3,000 ×
g
for 4 min at room tempera-
ture. Discard the supernatant and resuspend the pellet in
900
Preparation of Yeast
Competent Cells
l of 100 mM lithium acetate. Transfer the cell suspen-
sions to a 1.5 ml microcentrifuge tube. The cells are now ready
to be transformed with plasmid DNA (
see
Note 2
).
μ
1. For each transformation use 50
μ
l of competent cells and add
Yeast Transformation
in the indicated order: 240
l of 1 M
lithium acetate, and the transforming DNA (co-transformation
μ
l of 50 % PEG 4,000, 36
μ
