Applied Biosystems 7500 - Page 60

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
36
Notes
Design
Guidelines
When you design your own 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 reaction volume/well. Applied Biosystems recommends a reaction volume
of 25 µL for standard curve experiments. 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 (including units).
Note: For the Standard Concentration in Stock units 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