This document provides an optimized protocol for using the Stratagene Brilliant III Ultra-Fast QRT-PCR Master Mix with the 7900HT Fast Real-Time PCR System from Applied Biosystems. It includes detailed instructions for preparing the reactions.
The document outlines a step-by-step procedure for preparing reactions, including diluting the reference dye and combining reagent mixture components such as water, master mix, primers, probe, DTT, and RNA. It specifies suggested quantities for different RNA templates to achieve a final reaction volume of 20 μl.
| Final reaction volume | 20 μl |
|---|---|
| Probe concentration range | 100–600 nM |
| Primer concentration range | 200–600 nM |
| Total RNA quantity per reaction | 0.1 pg – 100 ng |
| mRNA quantity per reaction | 0.1 pg – 1 ng |
The final reaction volume should be brought to 20 μl.
You need to add 0.3 μl of diluted reference dye.