High Resolution Melt (HRM) Kit

Abbexa's High Resolution Melt (HRM) Kit has been developed for fast, highly reproducible HRM analysis and is validated on commonly used real-time instruments. The kit uses the latest developments in buffer chemistry and enhancers, and together with an antibody-mediated hot-start DNA polymerase system, ensures that this product delivers fast, highly-specific and ultra-sensitive HRM analysis.

This product is provided as a 2X master mix containing all the components for real-time PCR (qPCR), including the dye, dNTPs, stabilisers and enhancers. Only primers and template need to be added.

Target	High Resolution Melt (HRM) Kit
Tested Applications	PCR
Quality Control	The components of this kit have been extensively tested for activity, processivity, efficiency, heat activation, sensitivity along with absence of nuclease and nucleic acid contamination.
Storage	Store at -20°C. Avoid repeated freeze/thaw cycles.
Shelf Life	Up to 12 months.
Buffer	2X HRM Master Mix, containing dye, dNTPs, stabilisers and enhancers.
Kit Components	Reagent 500 × 20 µl (500 Runs) 2000 × 20 µl (2000 Runs) 2X HRM Mix 5 × 1 ml 20 × 1 ml
Assay Procedure	Preparing the PCR Master Mix The volumes given below are based on a standard 20 µl final reaction mix and can be scaled accordingly: Reagent Volume Final Concentration 2X HRM Mix 10 µl 1X 10 µM Forward Primer 0.8 µl 400 nM 10 µM Reverse Primer 0.8 µl 400 nM H2O up to 16 µl Total Volume 4 µl Suggested thermal cycling conditions The following conditions are suitable for 2 or 3 step qPCR cycles with amplicons of up to 200 bp and should be optimized to suit different machine specific protocols. It is not recommended to use annealing temperatures below 60°C or combined annealing/extension times longer than 30 seconds. 2-Step cycle: Cycles Temperature Time Notes 1 95°C 2 minutes (cDNA) 3 minutes (genomic DNA) Polymerase Activation 40 95°C 60-65°C 5 seconds *15-30 seconds Denaturation Annealing/Extension (acquire at end of step) 3-Step cycle: Cycles Temperature Time Notes 1 95°C 2 minutes (cDNA) 3 minutes (genomic DNA) Polymerase Activation 40 95°C 60-65°C 72°C 5 seconds 10 seconds *5-20 seconds Denaturation Annealing Extension (acquire at end of step) *It is not recommended to anneal/extend beyond 30 seconds. After the rection has reached completion, refer to the instrument instructions to complete the melt-profile analysis.
Availability	Shipped within 10-20 working days.
Note	THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC, THERAPEUTIC OR COSMETIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION.
Directions for use	General Considerations To prevent contamination, it is recommended that separate areas are used for PCR set up, PCR amplification and any post PCR gel analysis. Amplified PCR products should not be opened in the PCR set up area. Primers: Should have a melting temperature of 60°C. Optimal amplicon length should be 80-200 bp, and should not exceed 400 bp. A final primer concentration of 400 nM is suitable for most reactions, however it is recommended to perform a titration in the range of 0.1-1 µM to determine the optimal concentration. For optimal results use an equimolar primer concentration. Template: For genomic DNA templates, use up to 1 µg complex genomic DNA (such as eukaryotic) in a single PCR run. MgCl2: The MgCl2 concentration a 1X HRM mix is 3 mM. If needed, it is recommended to titrate MgCl2 to a maximum concentration of 5 mM. PCR Controls: To detect the presence of contaminating DNA, a no template control (NTC) should always be used, replacing the template with PCR-grade water.

Research Articles on High Resolution Melt (HRM) Kit

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