Chapter 3 Prepare the Relative Standard Curve Reactions
Prepare the Reaction Plate
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
60
Notes
Preparation
Guidelines
When you prepare your own relative standard curve experiment:
• If your experiment includes more than one target assay, prepare the reaction mix for
each target assay separately.
• Include excess volume in your calculations to provide excess volume for the loss
that occurs during reagent transfers. Applied Biosystems recommends an excess
volume of at least 10%.
• Include all required components.
• Prepare the reagents according to the manufacturer’s instructions.
• Keep the assay mix protected from light (in the freezer) until you are ready to use it.
Excessive exposure to light may affect the fluorescent probes.
•Prior to use:
– Mix the master mix thoroughly by swirling the bottle.
– Resuspend the assay mix by vortexing, then centrifuge the tube briefly.
– Thaw any frozen samples by placing them on ice. When the samples are thawed,
resuspend the samples by vortexing, then centrifuge the tubes briefly.
For More
Information
For more information on preparing the reaction mix, refer to the protocol appropriate for
the reagents you are using in the PCR reactions:
•TaqMan
®
Gene Expression Assays Protocol
• Custom TaqMan
®
Gene Expression Assays Protocol
Prepare the Reaction Plate
You prepare the reactions for each replicate group, then transfer them to the reaction plate
using the plate layout displayed in the 7500 software.
About the
Example
Experiment
For the relative standard curve example experiment:
•A MicroAmp
®
Optical 96-Well Reaction Plate is used.
• The reaction volume is 50 µL/well.
• The reaction plate contains:
– 12 Unknown wells
– 30 Standard wells
– 6 Negative control wells
– 48 Empty wells
• The plate layout that is automatically generated by the 7500 software is used:
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