UCP Multiplex PCR Kit
Ultra-Clean Production master mix for multiplex hot-start PCR and microbiome applications
The UCP Multiplex PCR Kit provides a convenient platform for highly-sensitive and specific multiplex hot-start PCR using any DNA or cDNA template.
Dedicated processes are implemented to enable Ultra-Clean Production of this master mix, including depletion of potential DNA background to a minimum. Combined with superior inhibitor resistance and minimized GC bias, this makes the kit an ideal choice for microbiome applications like 16S or 18S amplification. Additional features include: visual pipetting controls, a fast cycling protocol, room-temperature stability during and after reaction setup and a 4x concentrated master mix format, allowing for higher sample input volumes.
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Ultra-Clean Production processes combined with nucleic acid depletion ensure that potential contamination with residual DNA from expression hosts, or other sources, is reduced to a minimum. All components are tested for bacterial and fungal DNA, using generic PCR Assays for 16S and 18S rRNA coding sequences. This facilitates accurate results in microbial testing, quality control procedures or microbiome/metagenome analysis and sequencing. Particularly, testing low biomass samples will benefit from ultra-clean reagents, but also improved inhibitor resistance and minimal GC-content bias ensure accurate alpha- or beta-diversity results. The high-multiplexing degree, long-read capability, plus integrated visual pipetting and gel loading dyes, further improve efficiency in genotyping and genetic testing workflows.
The UCP Multiplex PCR Kit provides a convenient ready-to-use master mix formulation and UCP PCR water. It also contains orange UCP Master Mix Tracer and blue UCP Template Tracer in separate vials for optional use. (see figure ''UCP Multiplex PCR Kit''). The master mix includes a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers to the nucleic acid template, enabling high PCR efficiency of all targets (see figure "Unique PCR buffer"). Successful separation of 20 targets or more can be achieved by traditional gel or capillary electrophoresis, while sequencing-based detection strategies can accommodate significantly higher multiplexing degrees. At low temperatures, the DNA Polymerase is kept in an inactive state by an antibody and a novel Guard additive, which stabilizes the complex. (see figure ''Principle of Guard-protected hot-start mechanism''). This improves the stringency of the hot-start and prevents any enzymatic activity at ambient temperature, allowing reaction setup without ice or on instruments. The enzyme is fully activated after the 2-minute incubation step at 95°C and starts amplifying with high specificity from the first cycle. The hot-start procedure prevents extension from non-specifically annealed primers and primer-dimers in the first cycle ensuring highly specific and reproducible PCR. The blue and orange dyes in the UCP Template Tracer and in the UCP Master Mix Tracer, respectively, allow visual tracking of pipetted samples during the PCR setup, to prevent errors. When the blue template is added to the orange PCR Master Mix, the color changes to green, confirming that sample was added. (see figure ''Visual pipetting control''). The use of these tracers is optional. Reactions can be directly loaded onto agarose gels after cycling. Each tracer dye allows monitoring of the loading process and efficient tracking of the subsequent electrophoresis.
The UCP Multiplex Kit provides a convenient ready-to-use 4x concentrated master mix formulation, which allows a high-template input volume to further boost sensitivity.The orange Master Mix Tracer is added optionally to the master mix or PCR buffer. When template dyed with the blue Template Tracer is added to the master mix, the color of the solution changes from orange to green, providing a visual indication of correct pipetting and reaction setup (see figure ''PCR procedure''). The Tracer dyes allow direct loading on agarose gels after PCR, and efficient tracking of the subsequent electrophoresis. The dyes run at approximately 50 bp (orange) and 4000 bp (blue) on a 1% agarose gel (see figure ''Easy visualization''). The versatile buffer system allows ultrafast PCR in approximately 90 minutes or less, providing an additional protocol for 16/18S sequence amplification with low complexity. The Guard-protected hot-start mechanism enables complete reaction setup at room temperature, preventing the use of ice and allowing automated pipetting. To further streamline PCR workflows, we recommend the QIAgility instrument for automated reaction setup, and PCR analysis on the QIAxcel system. For microbiome analysis, QIAGEN offers a range of dedicated products including DNeasy Power Soil Pro Kits for sample extraction, QIAseq 16S/ITS panels for next-generation sequencing or CLC Microbial Genomics Pro Suite software for bioinformatic analysis.
The UCP Multiplex PCR Kit is ideally suited for procedures requiring a high purity and accuracy, like microbial testing, microbiome / metagenome analysis and sequencing, quality control, genotyping and genetic testing workflows.
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