HotStar PCR SuperMix provides qualified reagents for the amplification of nucleic acid templates by polymerase chain reaction (PCR). The mixture contains anti-Taq DNA polymerase antibody, Mg⁺⁺, dNTPs, and recombinant Taq DNA polymerase at concentrations sufficient to allow amplification during PCR. HotStar PCR SuperMix is supplied at 2X concentration to allow approximately 50% of the final reaction volume to be used for the addition of primer and template solutions. Reagents sufficient for 100 amplification reactions of 50 μl each are provided. Anti-Taq DNA polymerase antibody inhibits polymerase activity providing an automatic hot start and permits ambient temperature setup. Antibody-mediated hot starts improve PCR specificity and yield. Due to specific binding of the antibody, HotStar PCR SuperMix is present in an inactive form and is reactivated after a denaturation step in PCR cycling at 94°C. HotStar PCR SuperMix may be stored at either -20°C or 4°C. Storage at 4°C avoids the necessity of thawing the mix before assembling the PCR. Repeated freeze-thaw cycles might reduce performance or activity.
HotStar PCR SuperMix is evaluated in a DNA polymerization activity assay that measures the percent of Taq DNA polymerase inhibition versus an uninhibited control. A functional assay is also performed. Components of HotStar PCR SuperMix are tested for the absence of DNase, RNase and exonuclease activities. Recombinant Taq DNA polymerase is tested for the absence of exonuclease, and double- and single-stranded endonuclease activities.