Master mix for quantitative real-time PCR using labeled DNA probes
For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles, store dark
Storage at 4 °C for up to 3 months possible.
Shelf Life: 12 months
Concentration: 2x conc.
qPCR ProbesMaster with UNG/highROX is designed for the 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 Master 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 is achieved by an optimized hot-start polymerase. Its activity is blocked 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 contains UNG (Uracil-N-Glycosylase) and dUTP instead of dTTP to eliminate carry-over contamination of DNA from previous PCR reactions. The UNG treatment at the onset of thermal cycling removes uracil residues from dU-containing DNA and prevents it from serving as template.
The reaction chemistry of the kit is optimized for block-based PCR instruments that are compatible with the evaluation of the ROX reference signal.
qPCR ProbesMaster with UNG/highROX (red cap)
qPCR Pol, dATP, dCTP, dGTP, dUTP, UNG, 1 μM ROX, reaction buffer with KCl, (NH4)2SO4, MgCl2 and stabilizers
PCR-grade water (white cap)
ROX reference dye:
The qPCR ProbesMaster with highROX contains 500 nM ROX passive reference dye in the final assay. The dye does not take part in the PCR reaction but allows to normalize for non-PCR related signal variation and provides a baseline in multiplex reactions.
Dual-labeled DNA probes:
Real-time PCR technology based on dual-labeled DNA probes provides a high sensitive and high specific PCR system with multiplexing capability. It requires two standard PCR primers and the DNA probe that hybridizes to an internal part of the amplicon. The sequence of the dual-labeled DNA probe should avoid secondary structure and primer-dimer formation.
Preparation of the qPCR master mix:
The preparation of a master mix is crucial in quantitative PCR reactions to reduce pipetting errors. Prepare a master mix of all components except template as specified. 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.
|10 μl||25 μl||1x|
|0.6 μl||1.5 μl||300 nM|
|0.6 μl||1.5 μl||300 nM|
|0.4 μl||1 μl||200 nM|
|template DNA||x μl||x μl||<500 ng/assay|
|PCR-grade water||fill up to|
|fill up to|
Dispensing the master mix:
Vortex the master mix thoroughly to assure homogeneity and dispense the mix into real-time PCR tubes or wells of the PCR plate.
Addition of template DNA:
Add the remaining x μl of sample/template DNA to each reaction vessel containing the master mix and cap or seal the tubes/plate. Do not exceed 500 ng DNA per reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove possible bubbles.
Recommended cycling conditions:
|UNG treatment3)||50 °C||2 min||1x|
|95 °C||2 min||1x|
|Denaturation||95 °C||15 sec||35-45x|
|60-65 °C4)||1 min5)||35-45x|
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.