Power SYBR Green PCR Master Mix and RT-PCR Protocol (PN ...

Chapter 1
Introduction
Purpose of the Kit
About the Kit
The Power SYBR ® Green PCR Master Mix is a convenient premix of
the components (except primers, template, and water) necessary to
perform real-time polymerase chain reaction (PCR) using SYBR ®
Green I dye with enhanced sensitivity and specificity. The SYBR
Green dye binds to double-stranded (ds) DNA, thus providing a
fluorescent signal that reflects the amount of dsDNA product
generated during PCR.
You can perform One-Step or Two-Step RT-PCR using the Power
SYBR ® Green RT-PCR Reagents Kit (see “Materials and
Equipment” on page 1-4).
In RNA quantitation assays, you use the Power SYBR Green PCR
Master Mix in the second step of a Two-Step reverse-transcription
polymerase chain reaction (RT-PCR) protocol. In a One-Step RT-PCR
protocol, you add MultiScribe Reverse Transcriptase and RNase
Inhibitor to the Power SYBR Green PCR Master Mix.
You can use the Power SYBR Green PCR Master Mix with the
Applied Biosystems 7900HT Real-Time PCR System, the Applied
Biosystems 7500 Real-Time PCR System, the Applied Biosystems
7300 Real-Time PCR System, the ABI PRISM ® 7700 Sequence
Detection System (SDS), the ABI PRISM ® 7000 SDS, or the
GeneAmp ® 5700 SDS.
For the best quantitation results, use the following:
• Primer Express ® Software for primer design
• Applied Biosystems reagents
• Applied Biosystems universal thermal cycling conditions
Note: For optimal results, Applied Biosystems recommends
using the 9600 Emulation mode. However, using the Standard
(default) run mode with the Power SYBR Green PCR Master
Mix provides comparable results. Refer to the troubleshooting
section of the appropriate instrument user’s manual if you
encounter poor performance.
1-2 Power SYBR
DRAFT
® Green PCR Master Mix Protocol
September 29, 2005 1:41 pm, 1_Chapter.fm