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Jena BioscienceqPCR ProbesMaster from Jena Bioscience. Quantitative real-time analysis of DNA samples using DNA probe based detection.
Description: qPCR ProbesMaster is designed for quantitative real-time analysis of DNA samples using DNA probe based detection. The master mix is recommended for use with Dual Labeled Fluorescent Probes, e.
g.
TaqMan®, Molecular Beacons or FRET probes. It provides an easy-to-handle and powerful tool for quantification of sample DNA in a broad dynamic range of up to 6 orders of magnitude with exceptional sensitivity and precision.
The mix contains all reagents required for qPCR (except template, primer and labeled fluorescent probe) in a premixed 2x concentrated ready-to-use solution. The high specificity and sensitivity of the mix based on an optimized hot-start polymerase.
Its activity is blocked by antibody at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of nonspecifically annealed primers and primer-dimer formation at low temperatures during PCR setup.
The mix can also be used in combination with ROX reference dye (#PCR-351) in PCR instruments that are compatible with the evaluation of the ROX signal.
The sequence of the dual-labeled DNA probe should avoid secondary structure and primer-dimer formation.
A reaction volume of 20-50 μl is recommended for most real-time instruments. Prepare 13 volumes of master mix for 12 samples or a triple-set of 4 samples.
Pipet with sterile filter tips and minimize the exposure of the labeled DNA probe to light.
Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
component — 20 μl
assay — 50 μl
assay — final conc.
qPCR ProbesMaster — 10 μl — 25 μl — 1x
primer
forward
(10 μM)1) — 0.6 μl — 1.5 μl — 300 nM
primer
reverse
(10 μM)1) — 0.6 μl — 1.5 μl — 300 nM
dual-labeled probe
(10 μM)2) — 0.4 μl — 1 μl — 200 nM
template DNA — x μl — x μl — <500 ng/assay
PCR-grade water — fill up to
20 μl — fill up to
50 μl — -
1) The optimal concentration of each primer may vary from 100 to 500 nM. 2) Optimal results may require a titration of DNA probe concentration between 50 and 800 nM.
Tubes or plates should be centrifuged before cycling to remove possible bubbles.
Initial
denaturation and
polymerase activation — 95 °C — 2 min — 1x
Denaturation — 95 °C — 15 sec — 35-45x
Annealing and
elongation — 60-65 °C4) — 1 min5) — 35-45x
4) The annealing temperature depends on the melting temperature of the primers and DNA probe used. 5) The elongation time depends on the length of the amplicon.
A time of 1 min for a fragment of up to 500 bp is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters, especially of the annealing temperature may be necessary for each new combination of template DNA, primer pair and DNA probe.
