Before You Begin; Preparing The Pcr Master Mix - Applied Biosystems 7500 Getting Started Manual

Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System
STANDARD STANDARD STANDARD

Before You Begin

Before You Begin

Preparing the PCR Master Mix

Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
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 .
The second step (PCR) in the two-step RT-PCR procedure is amplifying the cDNA,
which you perform using the TaqMan
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.
Reaction Component
®
TaqMan Universal PCR
Master Mix (2✕)
Forward primer
Reverse primer
®
TaqMan probe
cDNA sample
Nuclease-free water
Total
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
are formulated so that the final concentration of the primers and probes are within the
recommended values.
®
Universal PCR Master Mix reagents.
µL/ Sample
Final Concentration
25.0 1✕
5.0 50 to 900 nM
5.0 50 to 900 nM
5.0 50 to 250 nM
5.0 10 to 100 ng
5.0 —
50.0 —
Universal Conditions
1✕
900 nM
900 nM
250 nM
—
—
®
Gene Expression Assays