Biomedical Engineering Reference
In-Depth Information
Primers for YC PCR reactions: The following primers can be used to check insert sizes in
both bait (pGBAD-B or pGBAE-B) or prey (pACTBD-B or pACTBE-B) vectors as they contain
common 5
and 3
recombination sequences.
h
Forward primer
:5
GAATTCACAAGTTTGTACAAAAAAGCAGGC 3
Reverse primer
:5
GTCGACCACTTTGTACAAGAAAGCTGGGTG 3
To avoid amplification of residual PCR insert from gap repair reactions, it is advisable to
use a combination of the forward primer above and a vector-specific reverse primer. This
is particularly important when screening colonies directly from gap repair plates.
Forward primer
:5
GAATTCACAAGTTTGTACAAAAAAGCAGGC 3
Reverse pGBAD-B or pGBAE-B primer
:5
GCCAAGATTGAAACTTAGAGGAG 3
Reverse pACTBD-B or pACTBE-B primer
:5
GTCGGCAAATATCGCATGCTTGTTC 3
•
Method
1 Pick a small amount of 5-10 colonies using a clean sterile toothpick for each
colony.
2 Dip the toothpick into 3
μ
lof0.02
M
NaOH. Do not remove all yeast. Resuspend the
remaining yeast in 20
μ
lofsterileH
2
O. These suspensions can be used for both
auto-activation and YC PCR (see below).
3 To preserve colonies, plate out 4
μ
l of yeast suspension onto selective plates and allow
to grow for 3-5 days.
4 To each of the yeast NaOH suspensions prepared in step 1 add 12
μ
l of the following
master mix. Amounts indicated provide enough master mix to perform 10
reactions.
Forward primer (10
μ
M
stock)
7.5
μ
l
Reverse primer (10
μ
M
stock)
7.5
μ
l
dNTPs: (10
μ
Mstock)
4.5
μ
l
NH
4
Buffer (10
×
)
15
μ
l
MgCl
2
(10 m
M
stock)
7.5
μ
l
DMSO
3
μ
l
d
H
2
O
73.5
μ
l
Taq polymerase
1.5
μ
l
5 PCR reactions should then be performed using the following program:
i. 95
◦
Cfor5min
ii. 95
◦
Cfor1min
iii. 55-68
◦
C for 1 min (dependent on primer Tm)
iv. 72
◦
C for 1-3min (use 1 min per Kb)
