For instrument compatibility refer to APPENDIX II PRODUCT DESCRIPTION: High Resolution Melt (HRM) analysis is a powerful technique for the analysis of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples. OUR MIX OFFERS: Accurate SNP genotyping Gene scanning CpG methylation analysis 3rd generation, saturating SuperGreen Efficient specific amplification from GC rich and AT rich sequences ADVANCED HRM Mix uses the novel SUPERGREEN intercalating dye does not inhibit PCR and is a saturating, dye essential for accurate SNP genotyping. SUPERGREEN can be used at high concentrations and does not have a different affinity for AT or GC rich sequences. Product Stability And Storage: On arrival store at -20°C. Avoid prolonged exposure to light. If stored correctly the mix will retain full activity for 12 months. It is stable at 4°C for 1 month and can go through 30 freeze/thaw cycles with no loss of activity. Instructions For Use: Note: For efficient amplification under fast cycling conditions we recommend amplicon lengths between 80bp and 200bp. Do not exceed 400 bp. The shorter the amplicon length the faster the reaction can be cycled. Primers should have a predicted melting temperature of around 60°C, using default Primer 3 settings. Reaction Setup: 1 Before starting, briefly vortex 2x WISENT ADVANCED HRM mix . 2 Prepare a final mix , recommended 20 ul, as follows:
Add
Volume
Final concentration:
WISENT ADVANCED HRM mix
10 ul
1X
Forward primer (10 uM)
0.8ul
400nM
Reverse primer (10 uM)
0.8ul
400nM
Template DNA
cDNA : < 100ng genomic DNA: <1ug
variable
Add PCR grade water up to 20 ul final volume
3 Program your instrument using following conditions, acquiring data on the SYBR® Green or FAM channel:
Cycles
Temperature
Time
Notes
1
95°C
cDNA:2min genomic DNA:3min
Polymerase activation
40
95°C
5 seconds
Denaturation
60°C to 65°C
20-30 seconds
Anneal/Extension, do not exceed 30 seconds, do not use temperatures below 60°C