Chapter 7 Prepare the Comparative C
T
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
144
Notes
Prepare the Reaction Mix
Prepare the reaction mix using the components and volumes that were calculated by the
7500 software (“Complete the Reaction Mix Calculations Tab for the Assays” on page 127).
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 when you prepare the reaction plate (see “Prepare
the Reaction Plate” on page 146).
About the
Example
Experiment
For the comparative C
T
example experiment:
• The reaction mix components are:
–TaqMan
®
Universal PCR Master Mix (2✕)
– 18S Assay Mix (20✕)
– EGR3 Assay Mix (20✕)
– MAOB Assay Mix (20✕)
– OGDH Assay Mix (20✕)
– OSGEP Assay Mix (20✕)
– SERPING1 Assay Mix (20✕)
–Water
• The volumes calculated in the software for the target assays are:
Note: The sample 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✕)2.50
Water 17.50
Total Volume 45.0
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