Biomedical Engineering Reference
In-Depth Information
8Add4
μ
l of a specific PCR reaction to each tube of suspended yeast and mix by
repeated pipetting. Also add 4
μ
lofH
2
O into one control tube to assess background.
This should be zero.
9 Use a PCR machine to incubate reactions at 30
◦
C for 30 min, followed by 25min at
42
◦
Cand1minat30
◦
C.
10 Add 100
μ
l of sterile water to each tube and mix well before spreading on selective
plates. SD low adenine media (- trp) for bait vectors or (- leu) for prey vectors.
Notes
d
Carrier DNA (salmon testis DNA) does not need to be sonicated. However, it must be heat
denatured (95
◦
C for 5 min) and placed on ice prior to use.
e
Genotype of the PJ69-4A host strain: MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4
gal80
LYS2::GAL1-HIS3, GAL2-ADE2 met2::GAL7-lacZ.
f
Tryptophan solutions should be
filter sterilized
(not autoclaved) and stored in the dark.
g
Cells should not be chilled during these procedures.
8.2.4 Identifying positive transformants
After 3 - 5 days' growth at 30
◦
C, colonies should be visible on transformation plates.
Pink/red colonies indicate the presence of an insert with an in-frame stop codon. White
colonies probably result from either auto-activating host cells or the presence of resid-
ual uncut vector. A selection of red colonies (around six) should be picked for each
construct and each colony should be analyzed to check insert size and the potential for
auto-activation.
8.2.5 Yeast colony PCR
The gap repair cloning methods described in Protocol 8.3 are directional. Therefore, inserts
should always be introduced with the correct orientation and reading frame. However, it
is important to ensure that the insert size is as expected. This can easily be checked using
primers that flank the recombination sites.
PROTOCOL 8.3 Using Yeast Colony PCR to Confirm Insert Size
Equipment and reagents
PCR tubes
•
PCR machine
•
Six positive colonies for each intended bait or prey construct
•
Sterile wooden tooth picks
•
