Catalogue Number: PCR-360S-JEN
|Shelf Life:||12 months|
|Shipping Condition:||Blue Ice|
|Unit(s):||2 x 1.25 ml|
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. 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.component||20 µlassay||50 µlassay||final conc. qPCR ProbesMaster||10 µl||25 µl||1x primerforward(10 µM)1)||0.6 µl||1.5 µl||300 nM primerreverse(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||