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qPCR Green Core Kit

Kit for real-time qPCR with green-fluorescent DNA stain

Product Cat. No. Amount Price (EUR) Buy / Note
qPCR Green Core Kit PCR-333S 100 reactions x 50 μl 90,00 Add to Basket/Quote Add to Notepad
qPCR Green Core Kit PCR-333L 500 reactions x 50 μl 360,00 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

Shelf Life: 12 months

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

Related products:

Description:
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.

Kit contents:
qPCR Pol (red cap)
5 units/μl

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.

component1x 50 μl
assay
13x 50 μl
assay
final conc.
qPCR GreenBuffer complete5 μl65 μl1x
dNTP Mix incl. dUTP1 μl13 μl200 μM
primer
forward
(10 μM)1)
1.5 μl19.5 μl300 nM
primer
reverse
(10 μM)1)
1.5 μl19.5 μl300 nM
UNG (PCR-353)2)0.2 μl2.6 μl0.2 units/assay
qPCR Pol0.25 μl3.25 μl0.025 units/μl
PCR-grade waterfill up to
40 μl
fill up to
520 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.

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 μl6 μl8 μl12 μl
final MgCl2 conc.2 mM3 mM4 mM6 mM

Dispensing the master mix:
Vortex the master mix thoroughly to assure homogeneity. Dispense 40 μl to a PCR tube or each well of the PCR plate.

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 °C2 min1x
Initial
denaturation and
polymerase activation
95 °C2 min1x
Denaturation95 °C15 sec30-40x
Annealing4)55-65 °C20 sec30-40x
elongation5)72 °C30 sec30-40x
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 used.
5) The elongation time depends on the length of the amplicon. A time of 30 sec 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 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