HRM qPCR
MasterMixes
HIGHLIGHTS
-
High-Resolution Melting Analysis (HRM)
-
Time and costs saving analysis of sequence variations
-
Universal - standard or fast cycling, GC or AT-rich templates
-
Highest sensitivity, no optimization required
-
Supplied with PCR Water for maximum convenience
APPLICATIONS
-
Detection of sequence variations
-
SNP genotyping
-
Methylation analysis
-
Mutation scanning
DESCRIPTIONS
High-Resolution Melting Analysis (HRM) is a fast and simple technique for the identification of DNA sequence variations. It allows identifying single nucleotide differences by detecting minor changes in qPCR melting curves HRM-qRT-PCR includes a proprietary intercalating saturating dye showing no inhibition for PCR. The dye has the same affinity for both AT or GC rich sequences what leads to the highest accuracy in genotyping. The hot-start function in the MasterMix is based on the small molecular inhibitor technology and allows achieving the highest sensitivity and specificity under both standard and fast PCR cycling conditions. The MasterMix provides excellent performance on both AT and GC rich templates and reliable results with minimum or no optimization.
COMPATIBILITY
Life Technologies: 7500, 7500 FAST, 7900, 7900HT FAST, 7900HT, Viia™7, QuantStudio™ 12K Flex
BioRad: CFX96™, CFX384™
Eppendorf: Mastercycler® ep realplex Mastercycler® realplex 2S
Illumina: Eco
QIAGEN: Rotor-Gene®Q, Rotor-Gene® 6000, Rotor-Gene® 3000
Roche Applied Science: LightCycler®480, LightCycler®96, LightCycler®Nano
PROTOCOL
Tips for successful workflow
-
Use special primer selection programs for good planning.
-
Work with amplicons in a range of 80-200, max 400 bp.
-
Take typical measures to prevent PCR cross-over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
-
Run reactions in triplets; include a no-template control and positive control in parallel.
-
Thaw and keep reagents on ice. Mix well before use.
-
Do not perform annealing/extension for more than 30 seconds and do not use lower than 60 °C temperature for this step.
To Prepare a 20 μl reaction, mix the following:
-
Reverse Primer: 100 - 400 nM final concentration
-
Forward Primer: 100 - 400 nM final concentration
-
Template
-
cDNA template <100
-
gDNA <1µg
-
-
Add PCR Water to 10 μl volume
-
InView qRT-PCR MasterMix (2X): 10 μl
-
Mix gently, avoid bubbles.
-
Place into the instrument and set onto SYBR® Green or FAM channel
PCR Program
-
Initial denaturation: 1 cycle: 95°C - 2 min for cDNA, 3 min for gDNA
-
Denaturation: 40 cycles: 95°C - 5 sec
-
Annealing/extension; 40 cycles: 60-65°C – 20-30 sec
Follow instrument instructions for melting curve analysis