Description
General description
FastStart TaqMan® Probe 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. It is a 2x concentrated master mix that contains all the reagents (except primers, probe, and template). This master mix simplifies the preparation of reactions for DNA detection and analysis. It allows very sensitive detection and quantification of defined DNA sequences.
Hot start protocols have been also been shown to significantly improve the specificity 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).
This FastStart TaqMan® Probe 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 a specific hydrolysis probe and PCR primers for each target.
Combination of this master mix with our Transcriptor First Strand cDNA Synthesis Kit achieves excellent results in two-step qRT-PCR.
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).
Application
The FastStart TaqMan® Probe Master has been used in:
quantitative, real-time DNA-detection assays
qPCR and two-step reverse transcription (RT)-qPCR
in the hydrolysis probe detection format
Features and Benefits
Increase qPCR sensitivity and specificity:
Produce lower cycle threshold (Ct) values with the hot start enzyme FastStart Taq DNA Polymerase and highest purity nucleotides included in the master mix.
Use it with any probe-based assay:
Achieve sensitive, specific results in assays with the Universal ProbeLibrary Probes or any other hydrolysis probe.
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.
Use with any real-time qPCR instrument other than the LightCycler® Instruments:
Choose from two formulations - one that contains the ROX reference dye and one without ROX.
Visualize amplification products on agarose gels.
Use robotic pipetting stations to set up qPCR reactions:
Benefit from a master mix that is stable at room temperature during extended reaction setup times.
Prevent carryover contamination:
The mix contains dUTP, so that it may be used with Uracil-DNA Glycosylase to prevent false positives arising from carryover contamination (i.e., contamination with amplified DNA).
Analysis Note
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).
Other Notes
qPCR targets
In principle, the FastStart TaqMan® Probe 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 and hydrolysis probes 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 TaqMan® Probe Master, 2x concentrated master mix that contains FastStart Taq DNA Polymerase, Reaction Buffer, and Nucleotides (dATP, dCTP, dGTP, dUTP).
For life science research only. Not for use in diagnostic procedures.
Legal Information
LightCycler is a registered trademark of Roche
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
