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Fast qPCR SybrMaster highROX

Fast master mix for real-time qPCR with SYBR® Green fluorescent DNA stain

Cat. No. Amount Price (EUR) Buy / Note
PCR-386S 2 x 1,25 ml (2x conc.) 149,20 Add to Basket/Quote Add to Notepad
PCR-386L 10 x 1,25 ml (2x conc.) 599,20 Add to Basket/Quote Add to Notepad

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 494 nm (bound to DNA), λem 521 nm (bound to DNA)

Description:
Fast qPCR SybrMaster highROX is designed to perform rapid real-time analysis of DNA samples.
The mix contains all reagents required for qPCR (except template and primers) in a premixed 2x concentrated ready-to-use solution. The mix generates results within 30-60 min and is recommended for routine PCR applications, high throughput PCR or genotyping. It provides an improved specificity and sensitivity when amplifying low-copy-number targets or working with complex backgrounds.
The fast amplification combined with high specificity and sensitivity is achieved with an optimized hot-start polymerase. Its activity is blocked by an 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 mix is recommended for use with PCR instruments that are compatible with the evaluation of the ROX signal.

SYBR® Green Fluorescent DNA Stain
SYBR® Green Fluorescent DNA Stain is a superior DNA intercalator dye specially developed for DNA analysis applications including real-time PCR (qPCR). Upon binding to DNA, the non-fluorescent dye becomes highly fluorescent while showing no detectable inhibition to the PCR process. The convenient master mix provides an easy assay set-up and handling for routine applications.
SYBR® Green is in contrast to EvaGreen® not recommended for high-resolution melting curve analysis (HRM).
To perform the SYBR® Green-based assay, select the optical setting for SYBR® Green on the detection instrument.

ROX reference dye:
Fast qPCR SybrMaster 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.

Content:
Fast qPCR SybrMaster highROX (red cap)
antibody-blocked fast hot start polymerase, dATP, dCTP, dGTP, dTTP, KCl, (NH4)2SO4, MgCl2, SYBR® Green DNA intercalator dye, 1 μM ROX, additives and stabilizers

PCR-grade water


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. 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.
Fast qPCR SybrMaster 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
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) Optimal results may require a titration of DNA probe concentration between 50 and 800 nM.

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 any bubbles.


Recommended cycling conditions:

Initial
denaturation and
polymerase activation
95 °C2 min1x
Denaturation95 °C1-10 sec35-45x
Annealing and
elongation
60-65 °C3)1-30 sec4)35-45x

3) The annealing temperature depends on the melting temperature of the primers and DNA probe used.
4) The minimal denaturation and elongation time mainly depends on the ramping speed oft the qPCR cycler. A denaturation time of 5 sec in combination with an elogation time of 10 sec is recommended for most cyclers. Further optimization may be required.

SYBR® is a registered trademark of Invitrogen Corporation, Carlsbad, California, USA

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