Biomedical Engineering Reference
In-Depth Information
3 Ideally, strong growth should be visible on plates lacking only tryptophan or leucine
but no growth should be visible on auto-activation plates.
j
4 Once non-auto-activating bait and prey clones have been identified, colonies can be
resuspended in SD media (
−
leu or - trp) containing 25% sterile glycerol and stored at
−
80
◦
C for future use. It is advisable to archive multiple clones of each construct.
k
Notes
i
Normally, the histidine antimetabolite 3-aminotriazole (3-AT) is added to (
−
his) media (at
2.5 mm) to reduce background growth. The concentration of 3-AT can be increased to eliminate
weak auto-activation on (
−
his) plates.
j
When auto-activation does occur, there are two typical phenotypes. If the bait or prey construct
induces auto-activation, strong growth will normally be seen across the entire area of the spot.
However, if auto-activation results from spontaneous mutations within the host, this will appear
as isolated colonies growing within the area of the spot. Examples of this type of background
growth can be seen in Figure 8.3c. If this kind of background occurs, individual colonies can
be isolated from the vector selection plate and retested for auto-activation. Alternately, a new
transformation can be performed using a fresh culture of host cells.
Some constructs may induce auto-activation on (
−
his) plates but not on more stringent
(
−
adeplates).Ifthisoccurs,itmaystillbe possible to perform screens under (
−
ade
selection). However, in this case it is important to also perform
β
Gal assays to ensure a second
independent reporter is being activated.
If either PJ69-4A or AH109 (MATa) haploid bait strains are mated with the Y187 (MAT
α
)
haploid prey strain the lacZ and HIS3 reporters cannot be scored independently as they both
have a common (GAL1) promoter.
k
Clones should not be maintained on selective plates for long periods to prevent the accumula-
tion of auto-activating mutations.
8.2.7 Targeted 'matrix'-style Y2H screens
The purpose of matrix-style Y2H assays (see Protocol 8.5 and 8.6) is to test for binary inter-
actions between a predefined set of bait or prey proteins. This approach can be performed
in a 96-well format using 12- or 8-tip multi-channel pipettes.
PROTOCOL 8.5 Performing Matrix Style Y2H Mating Assays
Equipment and reagents
•
96-well plate (round-bottomed or 'v'-shaped wells are best for this procedure)
•
Racked tips (1-20
μ
l and 1000
μ
l)
•
Multichannel pipettes (1-20
μ
l and 200
μ
l)
•
Sterile tooth picks
YPAD (rich media) plates
•
SD plates
−
trp
−
leu (diploid selection plates)
•
