Biomedical Engineering Reference
In-Depth Information
Circular filter paper (to fit selection plate)
•
Liquid nitrogen in a small insulated container.
•
Forceps
•
Sealable container
•
Z buffer: 60 m
M
Na
2
HPO
4
(8.5 g/l), 40 m
M
NaH
2
PO
4
(4.8 g/l), 10 m
M
KCl (0.75 g/l), 1 m
M
MgSO
4
(0.12 g/l)
•
Method
1Usingthe(
−
trp
−
leu) diploid selection plate prepared in Protocol 8.5 (step 15).
2 Incubate the plate for 4-5 days at 30
◦
C.
3 After the incubation, check the plate for even growth, then place a dry, round filter
paper onto the surface of the plate and apply even pressure to transfer cells onto the
paper.
4 Using forceps, slowly peel back the filter paper and then immerse in liquid nitrogen
(
∼
10 s). Repeat twice.
m
,
n
5 Place the filter paper (colony side up) into a Petri dish on top of two filter papers
which have been saturated with
β
-Gal assay mix (6 ml Z buffer, 100
μ
l of 100mg/ml
X-gal, 11
μ
l
β
-mercaptoethanol). 6ml of reaction mix is sufficient in a 150mm Petri
dish.
6 Incubate at 37
◦
C for 3-5 h until blue color develops.
o
,
p
Notes
m
Care should be taken at this stage as the filter may become brittle when frozen.
n
Care should also be taken when handling liquid nitrogen. Although only small quantities
are being used in this procedure, appropriate training is essential and institutional safety
procedures should be followed at all times.
o
The
β
-Gal assay mix is light sensitive and should be incubated in the dark.
p
As the
β
-Gal assay mix contains
β
-mercaptoethanol it is also advisable to make and dispense
solutions in a fume hood. Also, Petri dishes should be incubated in a well-sealed container.
8.2.7.1 Streamlining Y2H library screens
In many instances the function of a protein of interest may not be known. Also, most
researchers will not have access to large collections of individual prey clones. Therefore,
the only way to identify novel interaction partners is to screen high-complexity prey libraries.
Although this remains a valuable approach, library screening can be a daunting task. How-
ever, with a few simple adaptations, the process can be significantly simplified.
Historically, the main bottleneck in the process of library screening was the identifica-
tion of interacting prey inserts and the reconfirmation of observed interactions. Previously,
this process required the isolation of prey vectors from positive diploid colonies. Once iso-
lated, prey vectors were then reintroduced into fresh yeast, which would then be retested
