ROX

 Applied Biosystems 7900HT, 7500, 7700, 7000, StepOne™ & StepOnePlus™ Stratagene  Mx3000P™ & Mx3005P™

No ROX

Bio-Rad CFX96™ & CFX384™, iQ™5 & MyiQ™, Chromo4™, Opticon® 2 & MiniOpticon®; Qiagen  Rotor-Gene® Q, Rotor-Gene® 6000 Eppendorf Mastercycler® ep realplex² & ep realplex⁴

Description

HOT FIREPol® EvaGreen® qPCR Mix Plus is an optimized ready-to-use solution for real-time quantitative PCR assays, incorporating EvaGreen® dye. It comprises all the components necessary to perform qPCR: HOT FIREPol® DNA Polymerase, ultrapure dNTPs, MgCl2 and ROX dye according to system requirements. The user simply needs to add water, template and primers. EvaGreen® has several features such as high sensitivity, low PCR inhibition and room temperature stability that set it apart from other DNA-binding dyes used for quantitative real-time PCR applications. HOT FIREPol® DNA Polymerase is inactive at ambient temperatures and therefore requires an incubation step at 95°C for 15 minutes prior to qPCR. This prevents the extension of mis-primed products and primer-dimers formed at low temperatures during PCR setup.

EvaGreen® dye

• Highly sensitive – produces the most robust PCR signal when used at the recommended concentration
• Low PCR inhibition – exhibits much less PCR inhibition than other detection dyes via a smart "release on demand" DNA-binding technology
• Extremely stable – simply indestructible under most biochemical conditions. Can be stored at room temperature and be subject to repeated freeze-thaw cycles
• Spectrally similar to other popular dyes – compatible with all major brand real-time thermal cyclers
• Nonmutagenic and noncytotoxic

Features

• High sensitivity with broad dynamic range
• Unique stability at room temperature
• Based on EvaGreen® dye
• Convenient ready-to-use solution
• Reaction set-up without using ice
• Available with ROX reference dye and without ROX
• Mixes compatible with most real-time thermal cyclers

Applications

• Detection and quantification of DNA and cDNA targets
• Profiling gene expression
• Genotyping
• Microbial detection
• Viral load determination
• High Resolution Melt Analysis

Quality control

• Functional quality control via real-time PCR on different genomic templates
• Tests for stability at room temperature
• Tests for multiple freeze-thaw cycles

Performance data of EvaGreen® Mix

 Excellent sensitivity and specificity

The amplification of 98 bp fragment of GAPDH gene exhibits sensitive and efficient reaction curves (upper graph) with highly specific peak in melt curve analysis (lower graph) using HOT FIREPol® EvaGreen® qPCR Mix Plus (no ROX). Amplification was performed on human genomic DNA using Rotor-Gene® 6000 qPCR cycler following cycling protocols recommended by the suppliers.

High reproducibility on various qPCR cyclers

Twenty independent amplifications of each dilution show high uniformity and reproducibility on different qPCR platforms. The reactions were carried out on Corbett Rotor-Gene® 6000 (upper graph) using HOT FIREPol® EvaGreen® qPCR Mix Plus (no ROX) and Roche LightCycler 1.5 (lower graph) using HOT FIREPol® EvaGreen® qPCR Mix Plus (Capillary).