– Detection and quantification of DNA and cDNA targets
– Profiling gene expression
– Microbial detection
– Viral load determination
– Increased sensitivity and specificity for wide range of templates, including AT-rich, regular and GC-rich cDNA and gDNA
– Suitable for singleplex and duplex assays
– Reaction set-up at room temperature -master mix is stable at ambient temperature for one month
– Mix contains dUTP – it may be used with UNG to prevent carryover contamination from previous qPCR runs (UNG treatment)
– Use with any hydrolysis probe (LNA or DNA)
– Wide instrument compatibility: the mix is compatible with any real-time instrument other then capillary
– HOT FIREPol® DNA Polymerase
– 5x Probe Universal qPCR buffer
– 15 mM MgCl2 1x PCR solution – 3 mM
– Internal reference based on ROX dye
Remark for multiplex application: if ROX dye is used as one of the fluorophores, internal reference might interfere with the signal – version without ROX available upon request
qPCR performance in a duplex reaction: two genes from human gDNA were amplified in duplex reaction using HOT FIREPol Probe® Universal qPCR Mix. Excellent results were obtained from four 10x dilutions (starting from 10 ng/µl) from each gene. BAIP3 (blue) efficiency 100% and GAPDH (yellow) efficiency 98,4%. Reactions were performed on Applied Biosystems ViiATM 7 Real-Time PCR System.
Highly competitive qPCR mix: Five 10x dilutions of 197 bp long fragment of B4G4 gene with GC-content 75,6% were ampified from human gDNA using 5x HOT FIREPol® Universal qPCR Mix (blue) and qPCR Mix from another vendor (red). Reactions were performed on Applied Biosystems ViiATM 7 Real-Time PCR System following cycling protocol recommended by suppliers.
Internal reference based on ROX dye:
ROX is an internal passive reference dye used in some qPCR cyclers to normalize the fluorescent reporter signal generated in qPCR. It does not have any inhibitory effect on cyclers that do not need normalization.
Shipping and Storage Conditions:
Routine storage: -20ºC
Shipping and temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of 5x HOT FIREPol® Probe Universal qPCR Mix.