qPCR GreenMaster with highROX - clear

Master mix for real-time qPCR with green-fluorescent DNA stain

Product Cat. No. Amount Price (EUR) Buy / Note
S pack PCR-314S 2 x 1,25 ml (2x conc.) 110,00 Add to Basket/Quote Add to Notepad
L pack PCR-314L 10 x 1,25 ml (2x conc.) 440,00 Add to Basket/Quote Add to Notepad

Excitation (left) and emission (right) spectra of EvaGreen® bound to dsDNA in PBS buffer (pH 7.3).

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

Form: liquid

Concentration: 2x conc.

Spectroscopic Properties: λexc 500 nm (bound to DNA); λem 530 nm (bound to DNA)

Related products:

Description:
qPCR GreenMaster with highROX is designed for the quantitative real-time analysis of DNA samples using the fluorescent DNA stain EvaGreen®. The fluorescent dye in the master mix 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. It provides an easy-to-handle and 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 Master contains all reagents required for qPCR (except template and primer) in a premixed 2x concentrated ready-to-use solution. The high specificity and sensitivity of the mix 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 formations at low temperatures during PCR setup.
The 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 that are compatible with the evaluation of the ROX reference signal.
Kit contents:

  • qPCR GreenMaster with highROX(red cap)
    qPCR Pol, dATP, dCTP, dGTP, dUTP, EvaGreen, 1 μM ROX, reaction buffer with KCl, (NH4)2SO4, MgCl2 and stabilizers
  • PCR-grade water (white cap)

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.

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.
To perform the EvaGreen-based assay simply select the optical setting for SYBR® Green or FAM on the detection instrument.
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.

component20 μl
assay
50 μl
assay
final conc.
qPCR GreenMaster with highROX10 μl25 μl1x
primer
forward
(10 μM)1)
0.6 μl1.5 μl300 nM
primer
reverse
(10 μM)1)
0.6 μl1.5 μl300 nM
UNG
(PCR-353)2)
0.2 μl0.2 μl0.2 units/assay
template DNAx μlx μl<500 ng/assay
PCR-grade waterfill up to
20 μl
fill up to
50 μl
-

1) The optimal concentration of each primer may vary from 100 to 500 nM.
2) Only required if an UNG (Uracil-N-Glycosylase) treatment to prevent carry-over contaminations of DNA should be applied. UNG is not providet by this kit.

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:

UNG treatment3)50 °C2 min1x
Initial
denaturation and
polymerase activation
95 °C2 min1x
Denaturation95 °C15 sec35-45x
Annealing and
elongation
60-65 °C4)1 min5)35-45x

3) Cycling step 1 is only required if an UNG (Uracil-N-Glycosylase) treatment is applied.
4) The annealing temperature depends on the melting temperature of the primers and DNA probe used.
5) The elongation time depends on the length of the amplicon. A time of 1 min for a fragment of up to 500 bp is recommended.

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