Chapter 2
Design the Relative Standard Curve Experiment
Review the Reaction Setup
39
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
c.
Review the components and calculated volumes for the PCR reactions:
7.
In the Standard Dilution Series for GAPDH pane:
a.
Click the
Standard Concentration in Stock
field, then enter
100.0
.
b.
In the units drop-down list, select
ng
per
µ
L (default).
c.
Review the calculated volumes for preparing the standard dilution series:
Component
Volume (µL) for 1 Reaction
Master Mix (2.0
✕
)
25.0
Assay Mix (20.0
✕
)
2.50
Sample (10
✕
) or Standard
5.0
‡
‡ The sample or standard volume is limited to 10% of the total reaction volume.
H2O
17.50
Total Volume
50.0
1
2
3
5
4
6b
6a
6c
Dilution
Point
Source
Source
Volume
(µL)
Diluent
Volume
(µL)
Total
Volume
(µL)
Standard
Concentration
(ng/µL)
1 (200)
Stock
8.07
12.10
20.17
40.0
2 (20)
Dilution 1
2.02
18.15
20.17
4.0
3 (2)
Dilution 2
2.02
18.15
20.17
0.4
4 (0.2)
Dilution 3
2.02
18.15
20.17
0.04
5 (0.02)
Dilution 4
2.02
18.15
20.17
0.004
