This document provides an optimized protocol for using Stratagene Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix with QIAGEN Rotor-Gene Q Real-Time PCR Cycler. It includes instructions for preparing the reaction mixture and the suggested quantity range for different RNA templates.
The document serves as a quick reference guide for preparing reactions and setting up experiments using the Stratagene Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix. It details how to combine reagents, including nuclease-free water, primers, DTT, and RNA templates, into a final volume of 20 μl.
| Final reaction volume | 20 μl |
|---|---|
| Upstream primer concentration range | 150–500 nM |
| Downstream primer concentration range | 150–500 nM |
| DTT quantity per reaction | 0.2 μl of 100 mM DTT |
| Total RNA quantity range | 0.1 pg – 100 ng |
| mRNA quantity range | 0.1 pg – 1 ng |
| Number of cycles | 40 |
The protocol specifies bringing the final reaction volume to 20 μl.
The guide suggests using upstream and downstream primers at optimized concentrations between 150–500 nM.
The recommended quantity is 0.2 μl of a 100 mM DTT solution.
The suggested range is 0.1 pg – 100 ng for Total RNA and 0.1 pg – 1 ng for mRNA.