FastStart SYBR Green Master

FastStart SYBR Green Master General description

FastStart SYBR Green Master; Instructions For Use Ready-to-use hot start reaction mix without ROX for qPCR and RT-qPCR on real-time PCR systems other than the LightCycler®instruments. SYBR Green I is a DNA double-strand-specific dye.

During each phase of DNA synthesis, the SYBR Green I dye, which is included in the reaction mix, binds to the amplified PCR products; the amplicon can be detected by its fluorescence. Hot start protocols have been shown to significantly improve the specificity, sensitivity, and yield of PCR. Heat-labile blocking groups on some of the amino acid residues of FastStart Taq DNA Polymerase make the modified enzyme inactive at room temperature.

Therefore, there is no elongation during the period when primers can nonspecifically bind. The FastStart Taq DNA Polymerase is activated by removing the blocking groups at a high temperature (i.e., a pre-incubation step at +95°C).

The FastStart SYBR Green Master is a ready-to-use reagent mix that simplifies the preparation of reactions for qPCR and two-step qRT-PCR using the intercalating dye SYBR Green I for DNA detection and analysis. In combination with a real-time PCR instrument and suitable PCR primers, FastStart SYBR Green Master allows very sensitive detection and quantification of defined DNA sequences.

Do not use this product with the LightCycler® Instruments. In principle, the FastStart SYBR Green Master can be used for the amplification and detection of any DNA or cDNA target, including those that are GC- or AT-rich. However, you would need to adapt your detection protocol to the reaction conditions of the particular real-time PCR instrument used and design specific PCR primers for each target.

See the Operator′s Manual of your real-time PCR instrument for general recommendations.

Combine this master mix with our Transcriptor First Strand cDNA Synthesis Kit to achieve excellent results in two-step qRT-PCR. Insist on the Transcriptor First Strand cDNA Synthesis Kit – designed and function-tested for qPCR, and efficient with all real-time PCR instruments.

Application

The FastStart SYBR Green Master is a ready-to-use, 2x concentrated master mix that contains all the reagents (except primers and template) needed for running quantitative, real-time DNA detection assays[2], including qPCR[1] and two-step qRT-PCR, in the SYBR Green I detection format. Use the FastStart SYBR Green Master with any real-time qPCR instrument other than the LightCycler® Instruments.

Features and Benefits

• Improve PCR sensitivity and specificity: Rely on this mix′s FastStart Taq DNA Polymerase to minimize the formation of nonspecific amplification products through hot start PCR.

• Avoid over-estimation of qPCR results: Eliminate nonspecific amplification products and primer-dimers that would increase the amount of bound quantified SYBR Green I.

• Amplify and detect a broad range of DNA or cDNA targets: Amplify fragments up to 500 bp long, including those that are GC- or AT-rich. Works with any real-time PCR instrument other than the LightCycler® Instruments.

• Use any real-time PCR instrument other than the LightCycler® Instruments: Choose from two formulations – one that contains the ROX reference dye and one without ROX.

• Save time and effort in qPCR preparation: Rely on this easy-to-use 2x master mix to eliminate the need to mix components, titrate MgCl2, or perform other time-consuming optimization steps.

• Prevent false positives resulting from carryover contamination: The mix contains dUTP, so that it may be used with Uracil-DNA Glycosylase to eliminate contaminating DNA carried over from previous PCR reactions.

Components

FastStart SYBR Green Master, 2x concentrated master mix that contains FastStart Taq DNA Polymerase, Reaction Buffer, Nucleotides (dATP, dCTP, dGTP, dUTP), and SYBR Green I.

Quality Function test: Each lot is tested for performance in qPCR using three templates: a GC-rich template, an AT-rich template, and a long template (about 440 bp).

Other Notes

Cancer Research – Gene Expression Analysis You Can Count On Article (PDF, 622 KB) Gene Expression Analysis You Can Count On Article (PDF, 597.72 KB) qPCR targets In principle, the FastStart SYBR Green Master can be used for the amplification and detection of any DNA or cDNA target, including those that are GC- or AT-rich.

However, for each target, you would need: • to adapt your detection protocol to the reaction conditions of your real-time PCR instrument, and • design specific PCR primers for the target. See the Operator′s Manual of your real-time PCR instrument for general recommendations.

Two forms available The master mix is available in two forms – one that contains the ROX reference dye and one without ROX. Preventing carryover contamination This master contains dUTP, which will be incorporated into PCR products to help prevent false positives resulting from carryover contamination.

In subsequent PCRs, you can add Uracil-DNA Glycosylase to degrade any uracil-containing carryover contaminants (amplification products from previous PCRs). qRT-PCR Combine this master mix with our Transcriptor First Strand cDNA Synthesis Kit for two-step qRT-PCR.

This kit gives excellent results and works efficiently with all real-time PCR instruments. For life science research only. Not for use in diagnostic procedures.