I am trying to get HRM working on a corbett Rotor Gene 6000 using Qiagen Type-It Kit. For some unknown reason, I cannot get this machine to detect any amplification and subsequently there is nothing to melt during the HRM step. I have tried different reaction volumes and templates to try to get some amplification.

First reaction was 10ul total: 5ul 2X HRM mix, 0.7 Primer Mix, 4.3ul 1/50 gDNA dilution. --> this worked on a StepOne Plus machine but the software will not open for analysis. I'm trying to do this out of convenience for the lab.

Second reaction was 25ul total: 12.5ul 2X HRM mix, 1.75 Primer Mix, 6ul nuclease-free water, 4ul template (I tried 1/50 dilutions of zebrafish gDNA or 4ul of PCR product that was run on a gel and the concentration determined).

Reaction conditions:

95 degrees hold for 5 mins, 95 degrees for 10 secs, 50 degrees for 30 secs, 72 degrees for 10 secs (repeats steps 2-4 45 times). This was acquired using the green channel.

HRM stage: 50-95 degrees, measuring every 0.2 of a degree. This was acquired using the HRM A on HRM channel.

Both times I tried this, there was no amplification in the raw fluorescence graph. I know this reaction works with the reagents since it amplified and melted on a different machine. I'm not sure if there is something wrong with the machine or software.

I know the suggestion of using the other machine that I know works, might come up. For some reason that machine can register my amplification and I get nice clear melting profiles during the run but the HRM analysis software REFUSES to open my files to allow me to analyze them. So all I can have for rough estimates of indels is the melting curve I see during the run.