biotechrabbit™ 2X 5Star™ qPCR Probe Master Mix is an antibody-mediated hot-start real-time PCR mix optimized for amplification and quantification of viral DNA, cDNA and genomic DNA from a wide range of targets. The mix is tailored for high specificity and sensitivity in single and multiplex amplification; 5Star™ qPCR Mix is an excellent choice for the amplification of crude lysates, templates carrying PCR inhibitors and targets with low copy numbers.

5Star™ qPCR Probe Master Mix uses a proprietary enzyme and buffer formulation suitable for fast extension and multiplexing in challenging PCR reactions.

Notes

● For efficient amplification under fast cycling conditions, use amplicon lengths between 80 bp and 200 bp.
● Primers should have a predicted melting temperature of around 60 °C using default Primer3 settings (http://frodo.wi.mit.edu/primer3/).
● For TaqMan® probes, choose a probe close to the 5’ primer and avoid terminal guanosine residues.

Handling

Prevention of contamination

Contamination with undesired DNA is a concern when assembling the amplification reactions. To eliminate the possibility of contamination with undesired DNA, follow the guidelines below:

• Wear disposable gloves when handling the solutions.
• Dedicate separate sterile areas for the preparation of samples and reaction mixtures.
• Use molecular-grade nuclease-free water and reagents.
• Include a non-template control reaction in every PCR assay.
• Avoid carryover contamination.

Basic Protocol

• Calculate the required volume for your desired reaction numbers and consider one additional.
• Use high-quality PCR plates and seals designed for fluorescence applications.
• Reserve plate positions for positive (control DNA) and negative (water or buffer) controls.
• For 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