biotechrabbit™ UniFi™ 1-Step qRT-PCR Probe Master Mix provides outstanding performance for real-time PCR quantification of RNA templates, including mRNA, total RNA and viral RNA from a wide range of targets. The master mix ensures high specificity and sensitivity in single and multiplex detection, making it the ideal choice for extremely low-copy-number targets in pathogen detection.

The mix is provided as a 5X concentrated master mix, offering flexibility in pipetting schemes and template input for various PCR applications. UniFi™ 1-Step qRT-PCR Probe Master Mix uses proprietary reverse transcriptase technology and buffer chemistry for efficient cDNA synthesis and qPCR in a single tube.

Handling

Prevention of reaction contamination

RNase contamination is a particular concern when working with RNA. RNase A, which poses a major threat to RNA integrity, is a highly stable laboratory contaminant. To prevent RNA degradation and minimize the risk of contamination in one-step RT-PCR, follow the guidelines below:

• Use separate clean areas for preparation of samples and reaction mixtures.
• Use DEPC-treated tubes and pipette tips or certified nuclease-free labware with aerosol filters.
• Wear fresh gloves when handling RNA and all reagents.
• Assess RNA integrity before RT-PCR using denaturing agarose gel electrophoresis.
• Use only water and reagents that are free of DNA, DNases and RNases.
• With every one-step RT-PCR setup, perform a contamination control reaction without template RNA.

Basic Protocol

• Keep the master mix protected from light until use.
• Aliquot the master mix to minimize freeze-thaw cycles and light exposure.
• Thaw all reagents on ice and mix thoroughly. Assemble and keep all reactions on ice.
• Use only high-quality, optically clear reaction plates and seals designed for fluorescence applications.
• Do not use corner wells, or use a more robust seal.
• Reserve plate positions for positive controls (control RNA) and negative controls (water or buffer).
• First pipette the primer mixture, then add the template, and add the Master Mix last.
• Before preparing mixes, calculate the required volume based on the number of reactions plus one additional reaction.
• For better correlation, run the reactions in triplicate.

• Gently mix the reactions without creating bubbles. Do not vortex, as bubbles may interfere with fluorescence detection.
• Place the reaction into the PCR cycler.

Cycling program

*The recommended annealing/extension temperature is primer Tm +2 °C. Use gradient PCR to optimize the annealing temperature. Do not use annealing temperatures below 60 °C. For melt analysis, refer to the instrument instructions.

Product manual

Other documentation