Master mix for real-time qPCR with EvaGreen® fluorescent DNA stain
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.
Spectroscopic Properties: λexc 500 nm (bound to DNA), λem 530 nm (bound to DNA)
qPCR GreenMaster 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 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.
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.
qPCR GreenMaster (red cap)
antibody-blocked hot start polymerase, dATP, dCTP, dGTP, dTTP, KCl, (NH4)2SO4, MgCl2, EvaGreen® DNA intercalator dye, additives and stabilizers
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.
|qPCR GreenMaster||10 μl||25 μl||1x|
|0.6 μl||1.5 μl||300 nM|
|0.6 μl||1.5 μl||300 nM|
|template DNA||x μl||x μl||<500 ng/assay|
|PCR-grade water||fill up to 20 μl||fill up to 50 μl||-|
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:
|Initial denaturation and polymerase activation||95 °C||2 min||1x|
|Denaturation||95 °C||15 sec||35-45x|
|Annealing and elongation||60-65 °C3)||1 min4)||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.
EvaGreen® is a registered trademark and licensed for sale by Biotium, Inc., Hayward, CA, USA
SYBR® is a registered trademark of Invitrogen Corporation, Carlsbad, California, USA
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