Applied Biosystems 7500 - Review the Reaction Setup

Chapter 2 Design the Standard Curve Experiment
Review the Reaction Setup
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve
Experiments
32
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, standard dilution series, and
sample dilutions. If needed, you can edit the reaction volume, excess reaction volume,
component concentrations, standard concentration, and/or diluted sample concentration.
IMPORTANT! Perform the steps above for each target assay in the reaction plate.
About the
Example
Experiment
In the standard curve example experiment:
• Applied Biosystems RNase P assay is used.
• The reaction volume per well is 25 µL.
• The excess reaction volume is 10%.
• The reaction components are:
–TaqMan
®
Universal PCR Master Mix (2✕) or
TaqMan
®
2✕ Universal PCR Master Mix, No AmpErase
®
UNG
– RNase P Assay Mix (20✕)
– Sample or standard
–Water
• The standard concentration (stock) is 20,000 copies/µL.
• The diluted sample concentration is 6.6 ng/µL.
• The sample concentration (stock) is 100 ng/µL.
Complete the
Reaction Setup
Screen
Complete the Reaction Mix Calculations Tab (see page 33)
1. Select the Reaction Mix Calculations tab (default).
2. In the Select Target pane, select RNase P TAMRA (default).
3. In the Assay Type drop-down list, select Inventoried/Made to Order (default).
4. Make sure the Reaction Volume Per Well field displays 25 µL.
5. Make sure the Excess Reaction Volume field displays 10% (default).
6. In the Reactions for RNase P TAMRA pane:
a. Make sure the Master Mix Concentration field displays 2.0✕ (default).
b. Make sure the Assay Mix Concentration field displays 20.0✕ (default).