Biomedical Engineering Reference
In-Depth Information
— AP000555 (PRKM1, 255 bp)
◦
primer 3: 5
-TGGCTGATCTATGTCCCTGA-3
◦
primer 4: 5
-GCTCAGTTGTTTTGTGGGTAAG-3
— AC008575 (APC, 511 bp)
primer 5 GCTCAGACACCCAAAAGTCC
◦
primer 6: CATTCCCATTGTCATTTTCC
◦
•
PCR 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.
l
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 (5 ng/
μ
l)
m
•
0.1
μ
l of Amplitaq Gold DNA polymerase
•
5.45
μ
l of water.
3 Thermocycle as follows:
•
96
◦
C, 10 min
•
(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) TAE agarose gel electrophoresis.
n
Compare the
sizes of amplified products against molecular weight marker standards.
Notes
l
The multiplex PCR amplifies three amplicons, of 150, 255 and 511 bp. This method is comparable
to the van Beers method [40]. See Figure 3.1.
m
Use 10 ng of high molecular genomic DNA (e.g. prepared from freshly frozen tissue) as a
control.
n
DNA is considered to be of high quality if all three amplicons are visible on an agarose gel.
However, DNA samples showing both the 150 and 255 bp amplicons can be used for further
investigations.
