biotechrabbit CAPITAL qPCR Green Mix allows sensitive and specific amplification with an excellent signal to noise ratio and rapid extension rates. Extremely low-copy-number targets can be detected with high efficiency over several logs of template concentration, while primer-dimer formation is efficiently minimized.

CAPITAL qPCR Green Mix shows accurate and robust performance in wide range of applications, including gene expression and copy number variation analysis.

To enable the use of the kit on qPCR platforms with different reference dye concentration requirements, three kit formats are available: a 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 qPCR.

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.
• Amplicon lengths should not exceed 400 bp.
• Primers should have a predicted melting temperature of around 60°C, using default Primer 3 settings (http://frodo.wi.mit.edu/primer3/).

Prevention of PCR contamination

When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.

• Use separate clean areas for preparation of samples and reaction mixtures and for cycling.
• Wear fresh gloves. Use sterile tubes and pipette tips with aerosol filters for PCR setup.
• Use only water and reagents that are free of DNA and nucleases.
• With every PCR setup, perform a contamination control reaction that does not include 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

Product manual

Other documentation