Applied Biosystems 7500 Getting Started Guide

Chapter 5 Generating Data from RQ Plates – 7500 Fast System
Troubleshooting
56 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
FAST
FAST
FAST
Troubleshooting
Troubleshooting
Observation Possible Cause Action
High C
T
values/poor
precision or failed PCR
reactions
Target is difficult to amplify • Increase the annealing/extension time in the thermal
cycler protocol.
• Increase the annealing/extension temperature to 62 ° C.
Insufficient cDNA template is
present
Use 10 to 100 ng of cDNA template per 20-µL reaction.
Quality of cDNA template is poor 1. Quantify the amount of cDNA template.
2. Test the cDNA template for the presence of PCR
inhibitors.
Sample degradation Prepare fresh cDNA, then repeat the experiment.
The TaqMan 2✕ Universal PCR
Master Mix was used instead of
the TaqMan Fast Universal PCR
Master Mix (2✕)
, No AmpErase
UNG
Prepare the reactions with the correct Master Mix.
Primer-dimer formation To ensure optimal results, run the reaction plate as soon as
possible after completing the reaction setup. If you cannot
run a reaction plate within 2 hours after completing the
reaction setup, refrigerate or freeze the reaction plate until
you can load and run it on the 7500 Fast instrument.
Low ∆R
n
or R
n
values Extension time is too short Use the default thermal profile settings (see page 49).
Primer-dimer formation To ensure optimal results, run the reaction plate as soon as
possible after completing the reaction setup. If you cannot
run a reaction plate within 2 hours after completing the
reaction setup, refrigerate or freeze the reaction plate until
you can load and run it on the 7500 Fast instrument.
Run takes more than 40
minutes
Thermal cycler mode is set to
Standard or 9600 Emulation
Make sure that the thermal cycler mode is set to Fast (see
page 50).
Rn vs. Cycle plot is not
displayed
ROX dye was not selected as the
passive reference when the plate
document was set up
Select ROX dye as the passive reference when you set up
the plate document.
Extremely high ∆R
n
or
R
n
values
ROX dye was not selected as the
passive reference when the plate
document was set up
Select ROX dye as the passive reference when you set up
the plate document.
Evaporation Make sure that the reaction plate is sealed completely,
especially around the edges.

Applied Biosystems 7500 Specifications

General

Detection Method	Fluorescence
Sample Capacity	96-well plate
Thermal Cycler	Yes
Temperature Uniformity	±0.5°C
Type	Real-Time PCR System
Thermal Accuracy	±0.25°C
Detection Channels	5
Weight	45 kg
Power Requirements	100-240 V, 50/60 Hz
Dynamic Range	9 logs
Reaction Volume Range	10-100 µL
Temperature Range	4°C to 100°C
Thermal Cycler Block	96-well
Software	SDS Software
Detection	Multiplex (up to 5 colors)