Applied Biosystems 7300 Getting Started Guide

Chapter 5 Generating Data from RQ Plates – 7500 Fast System
Specifying Thermal Cycling Conditions and Starting the Run
48 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
FAST
FAST
FAST
Specifying Thermal Cycling Conditions and Starting the Run
Running Assays
Using Fast
Thermal Cycling
Conditions
Run assays using Fast thermal cycling conditions.
• Applied Biosystems has verified the performance of Fast thermal cycling and the
TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, for
quantitative applications only and not for endpoint applications, such as allelic
discrimination (SNP Genotyping or Plus/Minus Assays).
• Applied Biosystems has verified the performance of Applied Biosystems TaqMan
Gene Expression Assays and Custom TaqMan
®
Gene Expression Assays using the
default Fast thermal cycling conditions and the TaqMan Fast Universal PCR Master
Mix (2✕), No AmpErase UNG.
• It is expected that the vast majority of custom 5′ nuclease quantification assays
designed with the Applied Biosystems Assay Design Guidelines will provide
comparable performance when run using the default Fast thermal cycling
conditions and the TaqMan Fast Universal PCR Maser Mix (2✕), No AmpErase
UNG (as compared to running the standard thermal cycling conditions and the
TaqMan
®
2✕ Universal PCR Master Mix). If you encounter poor performance, see
“Troubleshooting” on page 56.
• When performing multiplex applications (when more than one target is amplified in
a single tube), it may be necessary to perform some assay reoptimization. Before
performing any multiplex applications, see the troubleshooting information on
page 56 for further information.
Expert Mode Expert mode allows you to select only those filters required for a particular experiment,
reducing run times to less than 30 minutes. Observe the following guidelines:
• The default thermal cycling protocol for Fast mode has an extension time of
30 seconds. This extension time has been tested for TaqMan Gene Expression
Assays, TaqMan Pre-Developed Assay Reagents and Primer Express software
designed assays that are run using default Fast thermal cycling mode conditions.
• Use of extension times below 30 seconds has been shown to affect the performance
of some assays. Validate the performance of assays with extension times of less
than 30 seconds.
• Applied Biosystems strongly recommends the use of ROX
™
dye to normalize
variation caused by pipetting error.
• When using the ROX passive reference feature, it is important to select both the
FAM
™
and ROX filters.
• Add extension time if you use more than three filters to allow for data collection
processes.
Note: The filters are labeled Filter A through E by default, but may be renamed as
desired. Select Tools > Filter Configuration to open the Filter Naming window.

Applied Biosystems 7300 Specifications

General

Brand	Applied Biosystems
Model	7300
Category	Laboratory Equipment
Language	English