Hey,
I would like to know more about HRM analysing. can anyone assist me regarding the analysing the data which were taken from this experiment?!
I have used MeltDoctor reagent and I calibrated quantstudio 3 with HRM plate in advance. Attached you can see the result from my samples.
Thank you in advance.
Fatemeh
Hello Fatemeh,
From the Image, it's really hard to depict the analysis since they're aligning to the negative scale and eliminating background noise will result to almost all samples being eliminated.
First Option:
I would suggest doing a normal amplification without the hrm master mix i.e., using the other taq polymerase Master mixes then the products after amplification, use: for 5X HRM master mix
HRM master mix 4.0ul
Nuclease free water 4.0ul
Template 2.0ul
Total 10.0ul
Then choose the HRM option alone (not the Pre-PCR with HRM option) if your machine has both options that is,
It should take like 20-30 minutes to complete the run.
Second Option,
You can try checking if the channels are actively acquiring during the PCR steps and also if the machine default dye is (ROX) some scenarios the master mixes and machine defaults defer resulting to ambiguous graphs.
I'll attach an image and publications I have assisted in carrying out HRM analysis to also assist.
Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance | Scientific Reports (nature.com)
Putative bloodmeal sources in Glossina austeni tsetse fly of Arabuko Sokoke National Reserve in Kenya | PLOS ONE
Thank you Tevin for your nice and useful comment, I will try to do the first option, I guess maybe it works more and it‘s more familiar to me.
thank you so much again for putting time to explain for me.
How big is your HRM amplicon? And what are you targetting? A single SNP? Methylation?? From the melting curves, it looks like your amplicons might be quite large ??
https://www.dna-utah.org/ is also helpful for HRM related content/software.
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