Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System
Specifying Thermal Cycling Conditions and Starting the Run
30 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
STANDARD
STANDARD
Specifying Thermal Cycling Conditions and Starting the Run
Default Thermal
Cycling
Conditions for
PCR
If you selected the two-step RT-PCR method for your RQ experiment (recommended),
you have already completed the RT step and are ready to PCR amplify cDNA.
The default thermal cycling conditions for the PCR step of the procedure, shown in the
following table, should appear in the Instrument tab.
Times and Temperatures (Two-step RT-PCR)
1) RT Step
HOLD HOLD * For reference only.
RT is complete at this point.
10 min @ 25 ° C 120 min @ 37 ° C
2) PCR Step
Initial Steps PCR (Each of 40 cycles)
AmpErase
®
UNG
Activation
AmpliTaq Gold
®
DNA
Polymerase Activation
Melt Anneal/
Extend
HOLD HOLD CYCLE
2 min @ 50 ° C 10 min @ 95 ° C 15 sec @ 95 ° C1 min @ 60° C
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