Fast master mix for quantitative real-time PCR using labeled DNA probes
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.
Fast qPCR ProbesMaster highROX is designed to perform rapid real-time analysis of DNA samples using DNA probe based detection. The master mix is recommended for use with Dual Labeled Fluorescent Probes, e.g. TaqMan®, Molecular Beacons or FRET probes.
The mix contains all reagents required for qPCR (except template, primer and labeled fluorescent probe) in a premixed 2x concentrated ready-to-use solution.
Amplification within 30-60 min 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.
ROX reference dye:
Fast qPCR ProbesMaster 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.
Dual-labeled DNA probes:
Real-time PCR technology based on dual-labeled DNA probes provides a high sensitive and specific PCR system with multiplexing capability. It requires two standard PCR primers and the DNA probe that hybridizes to an internal part of the amplicon. The sequence of the dual-labeled DNA probe should avoid secondary structure and primer-dimer formation.
Fast qPCR ProbesMaster highROX (red cap)
antibody-blocked fast hot start polymerase, dATP, dCTP, dGTP, dTTP, KCl, (NH4)2SO4, MgCl2, 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 ProbesMaster highROX||10 μl||25 μl||1x|
|0.6 μl||1.5 μl||300 nM|
|0.6 μl||1.5 μl||300 nM|
|0.4 μl||1 μl||200 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|