FastStart SYBR Green Master

FastStart SYBR Green Master; Instructions For Use

FastStart^™ SYBR^® Green Master is a ready-to-use hot start reaction mix without ROX for quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR on real-time PCR systems other than the LightCycler^® instruments. This master mix simplifies the preparation of reactions for DNA detection and analysis. In combination with a real-time PCR instrument, suitable PCR primers, and a hydrolysis probe, FastStart^™ TaqMan^® Probe Master allows very sensitive detection and quantification of defined DNA sequences.
SYBR^® Green I is a DNA double-strand-specific dye. During each phase of DNA synthesis, the SYBR^® Green I dye, 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 non-specifically 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 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.
Combine this master mix with our Transcriptor First Strand cDNA Synthesis Kit to achieve excellent results in two-step qRT-PCR.

The FastStart^™ SYBR^® Green Master has been used in qPCR and two-step qRT-PC in the SYBR^® Green I detection format. It is also used:

• in quantitative polymerase chain reaction (qPCR) for adeno-associated virus (AAV) titre quantification
• in qPCR to determine the expression level of different Col6a1-3 genes relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
• in quantitative real-time polymerase chain reaction (qRT-PCR) of G protein-coupled estrogen receptor-1 (GPER) mRNA
• in qRT-PCR for gene expression studies

Use the FastStart^™ SYBR^® Green Master with any real-time qPCR instrument other than the LightCycler^® Instruments.

• 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.

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).

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.

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.

For life science research only. Not for use in diagnostic procedures.

FastStart is a trademark of Roche

LightCycler is a registered trademark of Roche

SYBR is a registered trademark of Thermo Fisher Scientific or its subsidiaries

TaqMan is a registered trademark of Roche Molecular Systems, Inc.