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An easy and accurate way to determine the activity of a DNA polymerase without using radioisotopes.
EvaEZ™ Fluorometric Polymerase Activity Assay Kit provides an easy and accurate way to determine the activity of a DNA polymerase without using radioisotopes.
In the presence of DNA polymerase activity, the primer will be extended to form a double stranded product that can bind EvaGreen® Dye, resulting in an increase of fluorescence. The rate of increase of fluorescence is positively correlated to the activity of polymerase. The activity assay can be conducted at temperatures from 4°C to 75°C.
EvaEZ™ Polymerase Activity Mix is provided at 2X concentration with 5 mM MgCl2 (final Mg2+ concentration in assay is 2.5 mM). Magnesium-free EvaEZ™ Polymerase Activity Mix (29051MF) is available on request for customers who wish to control the magnesium concentration in the assay. Contact customer service at order@biotium.com to inquire about availability.
The ROX concentration is 35 nM which is suitable for low ROX instruments. If you are using a high ROX instrument, you can add ROX, 25 mM in TE Buffer (Catalog # 29052) which is available separately (Cat # 29052).
Our original EvaGreen® Dye is a saturating dsDNA binding dye that is superior for quantitative real-time PCR (qPCR), high-resolution melt (HRM), digital droplet PCR (ddPCR) and other genomics applications over SYBR® Green I and other commercial PCR dyes. It offers several essential features critical for PCR and related applications including high thermal, chemical and photostability, high sensitivity due to high signal to noise related to its novel release-on-demand mechanism, non-inhibitory to PCR, and lack of dye migration. In addition, EvaGreen® Dye is non-toxic, non-mutagenic, and not hazardous to aquatic life.
EvaGreen® Plus Dye is an advanced version of the original EvaGreen® Dye, retaining the same essential benefits while providing a higher signal-to-noise ratio. This greater sensitivity offers further advantages for digital PCR and isothermal applications.
EvaGreen® Dye is used in all of Biotiums dye-based qPCR master mixes, which combine superior brightness and sensitivity, with the ability to do melt curve analysis in the same reaction. The new EvaGreen® Plus Dye is available as a stand-alone 20X solution in water.
0.5X to 1X EvaGreen® is commonly used for LAMP applications when fluorescent signal is read by instrumentation. For detection by eye, an increased concentration of EvaGreen® can be used. For example, Lee, S. H. et al. tested up to 100X EvaGreen® and found that 10X EvaGreen® was optimal for their assay.
Lee, S. H. et al. Front. Microbiol., 09 January 2017 | https://doi.org/10.3389/fmicb.2016.02166