Chapter 2
Design the Relative Standard Curve Experiment
Review the Reaction Setup
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
38
Notes
7.
In the Standard Dilution Series for c-myc 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:
To complete the Reaction Mix Calculations tab
for the GAPDH assay
:
1.
In the Reaction Setup screen, select the
Reaction Mix Calculations
tab (default).
2.
In the Select Target pane, select
GAPDH
.
3.
In the Assay Type drop-down list, select
Inventoried/Made to Order
.
4.
Make sure
the Reaction Volume Per Well field displays
50 µL
.
5.
Make sure
the Excess Reaction Volume field displays
10%
.
6.
In the Reactions for GAPDH pane:
a.
Make sure the Master Mix Concentration field displays
2.0
✕
.
b.
Make sure the Assay Mix Concentration field displays
20.0
✕
.
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 (4)
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
7b
7a
7c
