Biomedical Engineering Reference
In-Depth Information
Table 2
Protocol for Preparation of Polymerase Chain Reaction Master Mix
Volume for one
Volume for
N
reaction (
µ
L)
reactions (
µ
L)
10X buffer A
5
5
×
N
MgCl
2
(25 m
M
)
8
8
×
N
dNTP mix
a
4
4
×
N
Forward primer (10 pmol/
µ
L)
5
5
×
N
Reverse primer (10 pmol/
µ
L)
5
5
×
N
MGB probe (2.5 pmol/
µ
L)
1
1
×
N
Uracil-
N
-glycosylase (1 U/
µ
L)
0.5
0.5
×
N
DMSO 100%
2.5
2.5
×
N
Ampli
Taq
Gold (5 U/
µ
L)
0.4
0.4
×
N
H
2
O
22.6
22.6
×
N
Total
N
a
dNTP mix: dATP, dCTP, dGTP at 50 µ
M
; dUTP at 100 µ
M
. MGB, minor groove-binding;
DMSO, dimethylsulfoxide.
45
45
×
Table 3
Thermal Profile of Real-Time
Polymerase Chain Reactions
Carried Out in an Applied
Biosystem 7700 Sequence Detector
50°C for 2 min
95°C for 1 min
95°C for 30 s
58°C for 1 min
72°C for 1 min
}
50 cycles
EBV DNA concentrations differed by up to six orders of magnitude in different
patients, the plots are compressed even with a logarithmic scale and the size
distribution of plasma EBV DNA in these individuals could not be easily ap-
preciated. To overcome this problem, the fractional concentration of each
amplicon was calculated by dividing its absolute concentration by the absolute
concentration determined by the assay with the shortest amplicon (82 bp). In
this way, size distribution of DNA molecules from different patients can be
summarized and compared.
Figure 2B
shows the plot of fractional concentra-
tions against amplicon lengths. There is a sharp drop of DNA concentrations
when the amplicon size is increased from 82 to 181 bp. On the other hand, the
drop in fractional concentration beyond the amplicon size of 181 bp is gradual.
