Fast master mix for real-time qPCR with SYBR® 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
Storage at 4 °C for up to 3 months possible.
Shelf Life: 12 months
Concentration: 2x conc.
Spectroscopic Properties: λexc 494 nm (bound to DNA), λem 521 nm (bound to DNA)
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.
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
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.
|Fast qPCR SybrMaster highROX||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|
|fill up to|
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:
|95 °C||2 min||1x|
|Denaturation||95 °C||1-10 sec||35-45x|
|60-65 °C3)||1-30 sec4)||35-45x|
SYBR® is a registered trademark of Invitrogen Corporation, Carlsbad, California, USA