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
42
Notes
Design
Guidelines
When you design your own relative standard curve experiment:
• If you are using TaqMan reagents, select the type of assay you are using:
–Select Inventoried/Made to Order if you are using Applied Biosystems
TaqMan
®
Gene Expression Assays (Inventoried or Made to Order) or Applied
Biosystems Custom TaqMan
®
Gene Expression Assays.
–Select Custom if you are designing your own assays with Primer Express
®
software.
• 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.
• Include excess reaction volume to account for the loss that occurs during pipetting.
Applied Biosystems recommends an excess reaction volume of at least 10%.
• Review the reaction mix concentrations for each target: If needed:
– For TaqMan reagents, edit the master mix and assay mix concentrations.
– For SYBR Green reagents, edit the master mix, forward primer, and reverse
primer concentrations.
– For 1-step RT-PCR, edit the reverse transcriptase concentration.
• Review the reaction mix components for each target:
– If you are running Fast PCR reactions, make sure you use Fast master mix in the
PCR reactions.
– If you are running standard PCR reactions, make sure you use standard master
mix in the PCR reactions.
– For 1-step RT-PCR, make sure you include reverse transcriptase in the PCR
reactions and use a specific buffer.
• Review the standard dilution series calculations for each target. If needed, edit the
Standard Concentration in Stock field (including units).
Note: For the Standard Concentration in Stock field, you can select ng or µg in the
drop-down list or you can enter another unit in the field (for example, copies, IU,
[International Units], nmol, pg, and so on). The table is updated according to your
entry.
• Review the sample dilution calculations for each sample. If needed, edit the diluted
sample concentration (including units) and stock concentration.
For More
Information
For more information on:
• Completing the Reaction Setup screen – Open the 7500 Software Help by clicking
or pressing F1.
• Applied Biosystems assays – Refer to the:
– TaqMan
®
Gene Expression Assays Protocol
– Custom TaqMan
®
Gene Expression Assays Protocol
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