Catalogue Number: PCR-368S-JEN
|Shelf Life:||12 months|
|Shipping Condition:||Blue Ice|
|Unit(s):||2 x 1.25 ml|
Description: qPCR GreenMaster highROX is designed for quantitative real-time analysis of DNA samples including High Resolution Melting (HRM) curve analysis. The mix contains all reagents required for qPCR (except template and primers) in a premixed 2x concentrated ready-to-use solution. It is recommended for routine PCR applications, high throughput PCR or genotyping and provides an improved specificity and sensitivity when amplifying low-copy-number targets or working with complex backgrounds. The mix is 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 fluorescent DNA stain EvaGreen® intercalates into the amplification product during the PCR process and allows the direct quantification of target DNA without the need to synthesize sequence-specific labeled probes (i.g. TaqMan® Probes). The reaction chemistry of the kit is optimized for instruments that are compatible with the evaluation of a high ROX reference signal. High Resolution Melting (HRM) analysis is a powerful tool for detection of mutations, polymorphisms and epigenetic differences in DNA samples. It is a fast and cost effective alternative to other genotyping technologies. EvaGreen® Fluorescent DNA Stain: EvaGreen® Fluorescent DNA Stain is a superior DNA intercalator dye specially developed for DNA analysis applications including real-time PCR (qPCR) and high-resolution DNA melting curve analysis (HRM). Upon binding to DNA, the non-fluorescent dye becomes highly fluorescent while showing no detectable inhibition to the PCR process. The dye is extremely stable both thermally and hydrolytically, providing convenience during routine handling. To perform the EvaGreen-based assay simply select the optical setting for SYBR® Green or FAM on the detection instrument. ROX reference dye: The qPCR GreenMaster 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.
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 GreenMaster with highROX||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 template DNA||x µl||x µl||