Biomedical Engineering Reference
In-Depth Information
The melting temperature of a DNA molecule depends on both its size and its nucleotide
composition; hence, GC-rich amplicons have a higher
T
m
than those having an abundance
of AT base pairs. During melt curve analysis, the real-time instrument continuously monitors
the fluorescence of each sample as it is slowly heated from a user-defined temperature below
the
T
m
of the products to a temperature above their melting point. Fluorescent dye is released
upon melting (denaturation) of the double-stranded DNA, providing accurate
T
m
data for
every single amplified product. Melting peaks are calculated by taking the differential (the
first negative derivative,
−
d
F
/d
T
) of the melt curve. These peaks are analogous to the bands
on an electrophoresis gel and allow for the qualitative monitoring of products at the end
of a run. Short primer dimers will melt at lower temperature than longer, target amplicon
products.
PROTOCOL 6.6 qPCR Assay Using SYBR Green I Dye
as the Reporter System
Equipment and reagents
2
×
commercial qPCR master mix buffer, including dNTPs, thermostable DNA polymerase
and SYBR Green I dye
u
,
v
•
25m
M
MgCl
2
(if required for optimization)
•
10
μ
M
primers
•
Real-time thermocycler with melt curve analysis option.
•
Method
1 Make a master mix combining, for each cDNA sample, the reagents in the order shown
below:
•
6.5
μ
l of water
w
•
0.5
μ
l of each 10
μ
M
primer
x
•
12.5
μ
lof2
×
qPCR master mix buffer.
Mix gently by repeatedly pipetting up and down (making sure there are no air
bubbles).
2 Add 20
μ
lofmastermixto5
μ
l of each template.
y
3 Centrifuge briefly to ensure there are no bubbles, and place into a thermocycler.
4 Perform a three-step PCR reaction according to the following thermal profile:
95
◦
C, 10 min
1 cycle:
Activation
95
◦
C, 15 s
40 cycles:
Denaturation
Annealing
z
60
◦
C, 30 s
Collect data
72
◦
C, 30 s
Extension
Collect data
