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QuantiTect Probe PCR Kits
For real-time PCR and two-step RT-PCR using sequence-specific probes
QuantiTect Probe PCR Kits enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C. Buy Products
QuantiTect Probe PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Product Details
Performance
The QuantiTect Probe PCR Kit delivers sensitive and reliable results in combination with the QuantiTect Reverse Transcription Kit (see figure "High sensitivity and efficiency, and wide dynamic range"). The unique PCR buffer composition enables QuantiTect Probe PCR Kits to provide sensitive quantification of low-copy DNA targets, as well as accurate quantification over a wide linear range (see figures "High sensitivity and efficiency, and wide dynamic range" and "Wide dynamic range in two-step RT-PCR"). HotStarTaq DNA Polymerase further increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases (see figure "Highly specific amplification"). A major source of PCR contamination is the carryover of PCR products from previous reactions. Pretreatment with UNG prior to starting PCR ensures that any contaminating PCR products do not affect subsequent PCR. The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect Probe PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figures "Effective UNG digestion" and "Wide dynamic range in real-time PCR").
Principle
QuantiTect Probe PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of gDNA and cDNA targets using sequence-specific probes. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan® and other dual-labeled probes), FRET probes, and Molecular Beacons. QuantiTect Probe PCR Kits contain a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products. QuantiTect Probe PCR Master Mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.
Procedure
QuantiTect Probe PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and DNA template to the ready-to-use PCR master mix, and start the reaction (see flowchart "Two-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions. For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
Applications
QuantiTect Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets or quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Probe PCR Kit, which has been specially developed for fast cycling on these instruments. Specifications
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