Chapter 3 Prepare the Relative Standard Curve Reactions
Prepare the Reaction Mix
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
58
Notes
Prepare the Reaction Mix
You prepare the reaction mix using the components and volumes that were calculated by
the 7500 software (“To complete the Reaction Mix Calculations tab for the c-myc assay”
on page 37 and “To complete the Reaction Mix Calculations tab for the GAPDH assay”
on page 38).
Note: The software calculates the volumes for all components for the PCR reactions.
However, when you prepare the reaction mix in this section, include only the master mix,
assay mix, and water. Add the sample or standard when you prepare the reaction plate
(see “Prepare the Reaction Plate” on page 60).
About the
Example
Experiment
For the relative standard curve example experiment:
• The reaction mix components are:
–TaqMan
®
Universal PCR Master Mix (2✕)
– c-myc Assay Mix (20✕)
– GAPDH Assay Mix (20✕)
–Water
• The volumes calculated in the software for both target assays are:
Note: The sample or standard is not added at this time.
Required
Materials
• Microcentrifuge tubes
• Pipettors
• Pipette tips
• Reaction mix components (listed above)
• Centrifuge
Component
Volume (µL) for
1 Reaction
Master Mix (2.0✕)25.0
Assay Mix (20.0✕)2.5
Water
17.5
Total Volume 45.0
To Next Page
Loading...