Kit for real-time qPCR with green-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
Shelf Life: 12 months
Spectroscopic Properties: λexc 500 nm (bound to DNA); λem 530 nm (bound to DNA)
qPCR Green Core Kit is designed for the quantitative real-time analysis of DNA samples using the fluorescent DNA stain EvaGreen®. The fluorescent dye in the reaction buffer intercalates into the amplification product during the PCR process and enables the rapid analysis of target DNA without the need to synthesize sequence-specific labeled probes. The kit provides a 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 high specificity and sensitivity of the Core Kit is achieved by an optimized hot-start polymerase. Its activity is blocked 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 included dNTP mix contains dUTP instead of dTTP and allows an UNG (Uracil-N-Glycosylase) treatment at the onset of thermal cycling to prevent carry-over contaminations of DNA from previous PCR reactions.
The reaction chemistry of the kit is optimized for block-based PCR instruments. The kit can also be used with ROX reference dye in PCR instruments that are compatible with the evaluation of the ROX signal. In this case, the ROX dye should be added as 1x concentration to the PCR reaction.
qPCR Pol (red cap)
dNTP Mix incl. dUTP (white cap)
10 mM dATP, 10 mM dCTP, 10 mM dGTP, 20 mM dUTP
qPCR GreenBuffer complete (green cap) - 10x conc.
200 mM Tris-HCl (pH 8.5), KCl, (NH4)2SO4, 30 mM MgCl2, EvaGreen and stabilizers
qPCR GreenBuffer without MgCl2 (blue cap) - 10x conc.
200 mM Tris-HCl (pH 8.5), KCl, (NH4)2SO4, EvaGreen and stabilizers
MgCl2 Stock Solution (yellow cap)
25 mM MgCl2
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.
The high quantum yield, excellent stability and lowest inhibition toward PCR makes it the ideal fluorophore in real-time PCR applications and a superior replacement for the widely used SYBR® Green I dye.
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||1x 50 μl|
|13x 50 μl|
|qPCR GreenBuffer complete||5 μl||65 μl||1x|
|dNTP Mix incl. dUTP||1 μl||13 μl||200 μM|
|1.5 μl||19.5 μl||300 nM|
|1.5 μl||19.5 μl||300 nM|
|UNG (PCR-353)2)||0.2 μl||2.6 μl||0.2 units/assay|
|qPCR Pol||0.25 μl||3.25 μl||0.025 units/μl|
|PCR-grade water||fill up to|
|fill up to|
Optimization of MgCl2 concentration:
A concentration of 3.0 mM Mg2+ as provided by the 10x qPCR Buffer complete (green cap) is recommended for most applications. For an individual optimization use the reaction buffer without MgCl2 and add MgCl2 Stock Solution as shown in the table below.
50 μl assay
|MgCl2 Stock Sol.||4 μl||6 μl||8 μl||12 μl|
|final MgCl2 conc.||2 mM||3 mM||4 mM||6 mM|
Addition of template DNA:
Add 10 μl of sample template DNA to each reaction vessel containing 40 μl master mix and cap or seal the tube / plate. Do not exceed 500 ng DNA per 50 μl reaction as final concentration. Tubes or plates should be centrifuged before cycling to remove possible bubbles.
Recommended cycling conditions:
|UNG treatment3)||50 °C||2 min||1x|
|95 °C||2 min||1x|
|Denaturation||95 °C||15 sec||30-40x|
|Annealing4)||55-65 °C||20 sec||30-40x|
|elongation5)||72 °C||30 sec||30-40x|
For optimal specificity and amplification an individual optimization of the recommended parameters, especially of the MgCl2 concentration and the annealing temperature may be necessary for each new combination of template DNA and primer pair.
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