Probe Universal qPCR Mix


Suitable for ROX-dependent and ROX-independent qPCR cyclers

For in vitro use only

PACK SIZES : 1ml | 8ml | 20ml

Download Protocol

 

Description:

5x HOT FIREPol® Probe Universal qPCR Mix is optimized for real-time quantitative PCR assays and contains all the components necessary to perform qPCR, with the exception of template, primers, and probe. The qPCR Mix contains optimized components and HOT FIREPol® DNA Polymerase supplied in a proprietary reaction buffer that enables efficient amplification of AT-rich, regular and GC-rich (up to 75%) targets. HOT FIREPol® Probe Universal qPCR Mix is optimized for DNA/LNA hydrolysis probes based on the 5´–3´ exonuclease activity.
HOT FIREPol® DNA Polymerase is activated by a 10 min incubation step at 95°C. This prevents extension of non-specifically annealed primers and primer-dimers formed at low temperatures during qPCR setup.
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Description

Applications:

– Detection and quantification of DNA and cDNA targets

– Profiling gene expression

– Microbial detection

– Viral load determination

Benefits:

– 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

Mix Composition:

– HOT FIREPol® DNA Polymerase

– 5x Probe Universal qPCR buffer

– 15 mM MgCl2 1x PCR solution – 3 mM

– dNTPs blend containing dUTP/dTTP

– 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

 

Product Performance:

The unique composition of the qPCR mix supports robust amplification of broad range of targets from regular to GC-rich.

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.

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