abi 7500 - Review the Reaction Setup

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
36
Notes
Design
Guidelines
When you design your own relative standard curve experiment:
• Enter a number from 10 to 100 for the reaction volume/well. The 7500 system
supports reaction volumes from 20 to 100 µL. The 7500 Fast system supports
reaction volumes from 10 to 30 µL.
• Review the thermal profile:
– Make sure the thermal profile is appropriate for your reagents.
– If you are performing 1-step RT-PCR, include a reverse-transcription step.
If your experiment requires a different thermal profile, edit the thermal profile or
replace the run method with one from the Run Method library. The Run Method
library is included in the 7500 software.
For More
Information
For more information on:
• The Run Method library or on completing the Run Method screen – Open the
7500 Software Help by clicking or pressing F1.
• Using Advanced Setup – See “Advanced Setup Workflow” on page 204.
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.
About the
Example
Experiment
In the relative standard curve example experiment:
• Applied Biosystems TaqMan
®
Gene Expression Assays are used.
• The reaction volume per well is 50 µL.
• The excess reaction volume is 10%.
• The reaction components are:
–TaqMan
®
Universal PCR Master Mix (2✕)
– c-myc Assay Mix (20✕)
– GAPDH Assay Mix (20✕)
– Sample or standard
–Water
• The standard concentration (stock) is 100 ng/µL.
• The diluted sample concentration is 50 ng/µL.
• The sample stock concentration is 100 ng/µL.