Applied Biosystems 7300 Getting Started Guide

Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System
Before You Begin
22 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
STANDARD
STANDARD
STANDARD
Before You Begin
Check that background and pure-dye runs have been performed regularly to ensure
optimal performance of the 7300/7500 system. For more information about calibrating
the 7300/7500 system, refer to the Online Help located within the software by clicking
the .
Preparing the PCR Master Mix
The second step (PCR) in the two-step RT-PCR procedure is amplifying the cDNA,
which you perform using the TaqMan
®
Universal PCR Master Mix reagents.
The TaqMan Universal PCR Master Mix Protocol (PN 4351891) explains how to use the
reagents in the kit. The following table lists the universal assay conditions (volume and
final concentration) for using the master mix.
CHEMICAL HAZARD. TaqMan Universal PCR Master Mix
(2✕) No AmpErase UNG may cause eye and skin irritation. Exposure may cause
discomfort if swallowed or inhaled. Read the MSDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and gloves.
If you design probes and primers using Primer Express software, they must be optimized
to work with the universal assay conditions, using the volumes listed in the table above.
All TaqMan
®
Custom Gene Expression Assays and TaqMan
®
Gene Expression Assays
are formulated so that the final concentration of the primers and probes are within the
recommended values.
Reaction Component µL/ Sample Final Concentration Universal Conditions
Taq M an
®
Universal PCR
Master Mix (2✕)
25.0 1✕ 1✕
Forward primer 5.0 50 to 900 nM 900 nM
Reverse primer 5.0 50 to 900 nM 900 nM
Taq M an
®
probe 5.0 50 to 250 nM 250 nM
cDNA sample 5.0 10 to 100 ng
Nuclease-free water 5.0 — —
Total 50.0 — —

Applied Biosystems 7300 Specifications

General

Brand	Applied Biosystems
Model	7300
Category	Laboratory Equipment
Language	English