Description
The Anstart Taq-D qPCR Master Mix (2×, with UDG) is a ready-to-use, probe-based qPCR master mix designed for sensitive and contamination-resistant fluorescence quantitative PCR detection in single or multiplex DNA targets. This product provides a simple, rapid, and highly sensitive PCR detection system, enabling real-time fluorescence PCR amplification in 35–40 minutes.Core ComponentsThe master mix contains:dNTP Mix (including dATP, dTTP, dCTP, dGTP, and dUTP)Antibody-modified hot-start DNA polymerase (Anstart Taq-D)UDG (Uracil-DNA Glycosylase)Users only need to add primers, probes (or fluorescent dyes), and template DNA to perform fluorescence-based qPCR.UDG Contamination Control MechanismDuring the initial 50°C, 2 min incubation, UDG cleaves the N-glycosidic bond between uracil and the sugar-phosphate backbone in any contaminating PCR products containing dUTP, releasing free uracil.Subsequent 95°C, 2.5 min heat treatment inactivates UDG while further degrading the phosphodiester backbone, effectively eliminating carryover contamination from uracil-containing PCR products.UDG acts on both single- and double-stranded DNA but is inactive against RNA.Compatible InstrumentsABI 7500, Qiagen QiaQuant 5-plexFor other instruments not listed, this kit has not been fully validated. If required, please contact our technical support team for further assistance.Protocol1. Reagent Preparation (Reagent Setup Area)1.1 Thaw the master mix at -20 ± 5°C, equilibrate to room temperature, vortex briefly, and centrifuge at low speed for 15 sec before use.Recommendation: Aliquot PCR reagents to avoid repeated freeze-thaw cycles.1.2 Prepare the PCR reaction mix according to the table below:ComponentVolume/25µLVolume/50µL2× Anstart Taq-D qPCR Master Mix12.5 µL25 µLPrimer-probe Mix1 µL2 µLTemplates5 µL10 µLddH2OUp to 25 µLUp to 50 µLTotal25 µL50 µLNote 1: Primer and template volumes can be adjusted as needed.Note 2: For fast cycling programs, a 25 µL reaction volume is recommended.1.3 Mix gently by pipetting or brief vortexing, then centrifuge briefly to collect liquid at the tube bottom.1.4 Transfer PCR tubes to the thermal cycler and initiate the reaction.2. Sample Preparation (Sample Prep Area)Add the appropriate sample volume as required.3. PCR Amplification (Amplification Area)3.1 Preheat the instrument and verify performance.3.2 Load prepared PCR tubes into the instrument and record tube positions. Set fluorescence channels as needed.Recommended Cycling ProgramsStandard ProgramStepCyclesTemp.TimeData Acquisition1150℃2minNo2195℃2min 30sNo34594℃10sNo44555℃40sYes5125℃10sNoFast ProgramStepCyclesTemp.TimeData Acquisition1150℃2minNo2195℃30sNo34594℃5sNo44555℃15sYes5125℃10sNoFluorescence acquisition can be set for 2-step or 3-step PCR depending on experimental needs.Optimal primer concentration: Typically 0.2 µM (Tm ≥ 55°C).Adjust annealing temperature and primer concentration for optimal amplification efficiency