biotechrabbit CAPITAL 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 choice for extremely low-copy-number targets in pathogen detection.

CAPITAL 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. To enable the use of the kit on qPCR platforms with different reference dye concentration requirements, three kit formats are available: a one-step kit containing no ROX, as well as LRox and HRox versions containing ROX in the corresponding concentrations.

Info: Recommended annealing temperature is 2°C above primer Tm (use gradient PCR to optimize the annealing temperature).

Quality Control

Functional assay

Mix tested functionally in QRT-PCR.

ROX reference dye

• See PCR cycler instruction for recommended concentration of ROX passive reference dye.

Notes

• For efficient amplification under fast cycling conditions use amplicon lengths between 80 bp and 200 bp.
• The shorter the amplicon length the faster the reaction can be cycled. Use maximum 400 bp amplicons.
• Primers should have a predicted melting temperature of around 60°C, using default Primer 3 settings (http://frodo.wi.mit.edu/primer3/).
• For TaqMan® probes choose probe close to 5’ primer, avoid terminal guanosine residues.

Prevention of reaction contamination

RNase contamination is an exceptional concern when working with RNA. RNase A, providing most threat to RNA integrity, is a highly stable contaminant of any laboratory. To prevent RNA from degradation and to minimize possibility of contamination One Step RT-PCR; follow the guidelines below:

• Use separate clean areas for preparation of the samples and the reaction mixture.
• DEPC-treat all tubes and pipette tips or use certified nuclease-free labware with aerosol filters.
• Wear fresh gloves when handling RNA and all reagents.
• Always assess the integrity of RNA prior to RT-PCR in 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 DNA.

Basic Protocol

• Keep the master mix protected from light until you use it.
• Aliquot the master mix to minimize freeze-thaw cycles and light exposure.
• Thaw on ice and mix very well all reagents. 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 (control DNA) and negative (water or buffer) controls.
• First pipette the primer mixture, then add the template and last the Master Mix.
• Before preparing mixes, calculate the volume needed according to the reaction number plus one extra.
• To have a better correlation, run the reactions in triplets.

• Gently mix the reactions without creating bubbles (do not vortex). Bubbles will interfere with fluorescence detection. Place the reaction into the PCR cycler.

Cycling Program

*Recommended annealing/extension temperature is primer Tm +2°C. Use gradient PCR to optimize the annealing temperature. Do not use temperatures below 60°C. Do not exceed 30 seconds.
For melt analysis refer to instrument instructions. 

Product manual

Other documentation