Biomedical Engineering Reference
In-Depth Information
v. Repeat steps ii-iv 34-39 times
vi. 72
◦
Cfor5min
vii. Hold at 15
◦
C.
6 Run out 5-10
μ
l of each PCR product on a 1% agarose gel to determine the size of
individual PCR products.
Notes
h
If further verification of inserts is required, PCR products can be directly sequenced to generate
either sequence tags or full primary sequence, depending on insert size.
8.2.6 Bait and prey auto-activation tests
It is essential to test both bait and prey clones to ensure that they do not independently acti-
vate Y2H reporter genes (see Protocol 8.4). There can be several reasons for auto-activation.
Common causes include: the occurrence of spontaneous host mutations, which allow cells
to grow on selective media in the absence of bait/prey interactions; bait proteins that have
inherent transcriptional activity or are able to interact with other factors, which are capable
of activating transcription. Also, prey proteins that bind DNA may non-specifically drive
transcription of two hybrid reporter genes.
PROTOCOL 8.4 Testing for Auto-Activation of Bait and Prey Clones
Equipment and reagents
•
Tooth picks
•
SD (
−
trp
−
his + 2.5 m
M
3AT) plates and SD (
−
trp
−
ade) selective plates (for analysis
of bait constructs)
•
SD (
−
leu
−
his + 2.5 m
M
3AT) plates and SD (
−
leu
−
ade) selective plates (for analysis
of prey constructs)
•
Bait and prey clones containing vectors with verified size inserts.
Method
1 Spot 3
μ
l of the resuspended yeast from Protocol 8.3 (step 2) onto SD plates lacking
either tryptophan (baits) or leucine (preys).Atthesametime,spotthesamevolume
onto bait auto-activation plates, SD (
−
trp
−
his
+
2.5 m
M
3AT) plates and SD (
−
trp
−
ade) or SD (
−
leu
−
his
+
2.5m
M
3AT) and SD (
−
leu
−
ade) selective plates for
preys.
i
2 Incubate at 30
◦
C for an equivalent time to that being used to investigate potential
interactions. This may range from 7 to 14 days.
