Biomedical Engineering Reference
In-Depth Information
PROTOCOL 1.4 DNA Quality Control PCR
Equipment and reagents
•
Primer stocks (100
μ
M
):
—RS2032018 (150 bp)
◦
primer 1: 5'-GTGTCTCCCTTCCCACTCAA-3'
◦
primer 2: 5'-AGCCCACCTACCTTGGAAAG-3'
—AP000555 (PRKM1, 255 bp)
◦
primer 3: 5'-TGGCTGATCTATGTCCCTGA-3'
◦
primer 4: 5'-GCTCAGTTGTTTTGTGGGTAAG-3'
—AC008575 (APC, 511 bp)
◦
primer 5 GCTCAGACACCCAAAAGTCC
◦
primer 6: CATTCCCATTGTCATTTTCC
•
Polymerase chain reactions (PCRs) reaction buffer II without MgCl
2
(Applera)
•
Amplitaq gold DNA polymerase (5 units/
μ
l Applera).
Method
1 Prepare a primer working solution mix containing 20 pm/
μ
l of primers 1 and 2, and
10 pm/
μ
l for primers 3-6.
f
2 Prepare a 10
μ
l reaction by mixing the following:
•
0.25
μ
lofprimermix
•
1.0
μ
lof10
×
PCR reaction buffer II without MgCl
2
•
0.2
μ
lof4
×
10 m
M
dNTPs
•
1.0
μ
lof25m
M
MgCl
2
•
2.0
μ
l of template DNA (1-250 ng)
g
,
h
•
0.1
μ
l of Amplitaq gold DNA polymerase
•
5.45
μ
l of water.
3 Thermocycle as follows:
•
96
◦
C, 10min
•
(94
◦
C, 30 s; 55
◦
C, 30 s; 72
◦
C, 1 min)
×
35
•
72
◦
C, 5 min.
4 Analyze each PCR product by 2% (w/v) Tris-acetate-EDTA agarose gel electrophoresis.
Compare the sizes of amplified products against molecular weight marker
standards.
i
