Chapter 6
Design the Comparative C
T
Experiment
Review the Reaction Setup
127
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Review the Reaction Setup
In the Reaction Setup screen, select the assay type (if using TaqMan reagents), then
review the calculated volumes for preparing the PCR reactions and sample dilutions. If
needed, you can edit the reaction volume, excess reaction volume, component
concentrations, and/or diluted sample concentration.
About the
Example
Experiment
In the comparative C
T
example experiment:
• Applied Biosystems TaqMan
®
Gene Expression Assays are used.
• The reaction volume per well is 50
µ
L.
• The excess reaction volume is 10%.
• The reaction components are:
– TaqMan
®
Universal PCR Master Mix (2
✕
)
– Assay Mix (20
✕
) for each target (18S, EGR3, MAOB, OGDH, OSGEP, and
SERPING1)
– Sample
– Water
• The diluted sample concentration is 50 ng/
µ
L.
• The sample stock concentration is 100 ng/
µ
L.
Complete the
Reaction Setup
Screen
IMPORTANT!
Perform the following steps for each target assay in the reaction plate.
Complete the Reaction Mix Calculations Tab for the Assays
1.
Select the
Reaction Mix Calculations
tab (default).
2.
In the Select Target pane, select
18S
.
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 18S pane:
a.
Make sure the Master Mix Concentration field displays
2.0
✕
.
b.
Make sure the Assay Mix Concentration field displays
20.0
✕
.
