Biomedical Engineering Reference
In-Depth Information
18 Incubate plates at 30
◦
C for up to 10 days. Selective growth can normally be seen
within 3-5 days. However, weak interactions may take longer to develop
(Figure 8.3).
(a)
- trp/- leu
(b)
- trp/- leu/- his +3AT
(c)
- trp/- leu/- his +3AT
Figure 8.3
An example of a targeted matrix style Y2H study. In this study a bait clone
containing a human E3-RING protein has been mated against a collection of human
E2 Ubiquitin conjugating enzymes. (a) Growth of diploid yeast on (
−
trp
−
leu) plates
following mating of bait and prey clones. (b) Growth of the same colonies after transfer to
selective reporter plates (
−
trp
−
leu
−
his
+
2.5m
M
3AT). On these plates it is possible to
see increased growth of some colonies, indicating positive bait-prey interactions. (c) The
top section shows an example of a selective reporter plate showing signs of background
growth, which is typical of spontaneous mutations in the host yeast. Significantly, because
of the colony spotting method used in these screens, it is still possible to score positive
interactions above background. This would not be the case if diploids had been patched
out onto selective plates. In this case growth would have been more uniform throughout
the patch, thereby preventing accurate scoring of true positive interactions.
19 Record growth at regular intervals by photographing plates.
Notes
l
It is important to replicate onto (
−
ade) selection plates before (
−
his) plates. If (
−
his) plates
are used first, too much yeast may be transferred, producing high background growth, which
makes positive interactions harder to detect.
PROTOCOL 8.6 Performing
β
-Gal Reporter Assays
Equipment and reagents
Clean 150mm Petri dish
•
(
−
trp
−
his) diploid selection plates
•
