Biomedical Engineering Reference
In-Depth Information
In the methods described here, we assume that the reader is
screening a collection of mutant strains containing the GSS
reporter, which are arrayed on agar or in microtiter plates. The
array format is advantageous even when working with a small num-
ber of strains as it allows multiple replicates to be assayed under
identical conditions and provides highly sensitive, reproducible
data. Genome-scale yeast mutant collections are commercially
available from a variety of sources; the reporter can be introduced
to a mutant array by conducting a large-scale genetic cross using a
special query strain and synthetic genetic array (SGA) technology.
For a detailed description of the SGA methodology, which is
beyond the scope of this chapter, we refer the reader to Tong et al.
[
9
]. Below we describe how to introduce the reporter into a single
yeast strain, which could then be assayed directly or used as a start-
ing point for introducing the reporter into mutant arrays by SGA.
1. Double digest the GSS plasmid with NotI and SnaBI (
see
Note 10
). This releases a 6,100 bp fragment containing the
chimera as well as a 4,900 bp fragment corresponding to the
backbone vector.
2. Transform 1-3
3.1.1 Introducing
the GSS Reporter into
Yeast Strains
ʼ
g digested plasmid into desired yeast strains
(
see
Note 11
).
3. Select for integration of the construct by plating yeast on
YEPD + ClonNat (
see
Note 12
).
4. Incubate for 2-3 days at 30 °C.
5. Streak resulting transformants to obtain single colonies on
YEPD + ClonNat plates.
6. Test isolates for absence of growth on SD-URA plates to elimi-
nate colonies that contain the uncut plasmid.
7. Confi rm GSS has been integrated into the
SUC2
locus by col-
ony PCR or sequencing.
Once a collection of yeast strains that contains the GSS reporter
and mutations of interest is generated, a 96-colony array can be
made by patching yeast onto media in an 8 × 12 grid in a random
fashion. This 96-colony array will be the starting point for the
assay. For the sake of the protocol, we assume the mutants contain
a G418 resistance marker. If not, media containing G418 will have
to be changed accordingly.
Yeast arrays should be maintained as frozen stocks in 96-well
plates containing YPD +15 % glycerol. Liquid cultures frozen in
96-well plates should be pinned to agar in 96-density format before
condensing to make 384 stock arrays (
see
Note 13
). To access the
array from frozen stocks, the array is pinned from the liquid to a
solid agar plate. For ease of handling, the original 96-array plates
are condensed fourfold, producing a set of 384-density arrays to
give four biological replicates (
see
Notes 14
and
15
).
3.1.2 Replicating
96- and 384-Density
Arrays by Manual Pinning
