FastStart Universal SYBR Green Master (Rox)
Universal ready-to-use hot start reaction mix for qPCR and RT-qPCR on all real-time PCR systems requiring normalization with ROX.
For life science research only. Not for use in diagnostic procedures.
FastStart Universal SYBR Green Master (Rox) includes a novel reference dye that enables its use on all real-time PCR instruments requiring normalization with ROX, without modifications or adjustments to the specific instrument or protocol. This ready-to-use, 2x concentrated master mix contains all reagents (except primers and template) needed for running quantitative, real-time DNA detection assays, including qPCR and two-step qRT-PCR, in the SYBR Green I detection format. FastStart Universal SYBR Green Master (Rox) generates excellent results on instruments such as the Applied Biosystems 7900 HT Fast Real-Time PCR System or the Applied Biosystems 7500 Real-Time PCR System (see Figure 1). This product is not intended for use with LightCycler® Instruments.
Improve PCR sensitivity and specificity.
Minimize the formation of nonspecific amplification products.
Avoid over-estimation of qPCR results.
Eliminate nonspecific amplification products and primer-dimers that 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.
Save time and effort in qPCR preparation.
Eliminate the need to mix components, titrate MgCl2, or perform other time-consuming optimization steps.
Prevent false positives resulting from carryover contamination.
Use this dUTP-containing mix with Uracil-DNA Glycosylase to eliminate contaminating DNA carried over from previous PCR reactions.
FastStart Universal SYBR Green Master (Rox), 2x concentrated master mix that contains FastStart Taq DNA Polymerase, Reaction Buffer, Nucleotides (dATP, dCTP, dGTP, dUTP), SYBR Green I, and a reference dye.
FastStart Universal SYBR Green Master (Rox) can be used for the amplification and detection of any DNA or cDNA target, including those that are GC- or AT-rich.
Combine this master mix with Transcriptor First Strand cDNA Synthesis Kit (Roche) to achieve excellent results in two-step qRT-PCR.
Figure 1: Quantification of the human globin gene with the FastStart Universal SYBR Green Master (Rox) (a) and a master mix from another supplier (b) on an Applied Biosystems 7500 Real-Time PCR System. The FastStart Universal SYBR Green Master (Rox) shows higher sensitivity and more specific performance.
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 (+15 to +25°C). Therefore, there is no elongation during the period when primers can nonspecifically bind. FastStart Taq DNA Polymerase is activated by removing the blocking groups at a high temperature (i.e., a pre-incubation step at +95°C).
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 (approximately 440 bp).