biotechrabbit CAPITAL™ qRT-PCR Green Mix allows sensitive and specific cDNA synthesis and qPCR in a single tube for quantifying mRNA, total RNA and viral RNA sequences. Extremely low-copy-number targets can be detected with high efficiency over several logs of template concentration.

CAPITAL qRT-PCR Green 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 concentratios.

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/).

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