Hi Olivier,

I tried 2 different master mix: LifeTechnologies and Qiagen. Each mastermix works better for their own machines (Qiagen master mix works better in the Rotorgene). We tried to make HRM work for a whole year in the 7500Fast Real time (Applied Biosystems) but it didn't work. The first time that I tried in the Rotorgene, it worked very well.

In my experience, the mixes is not problem. But the system. See the sensibility require to detect the diferents melting curves. If you see the techical information of the machines you can just to see that Life machine does not work for small change in the sequence. It is because the range of temperature modification is not good.(the software for HRM is not good because the change in temperaturemeltin is sensitive enough) You need work with system that can change in 0.01 temperature interval. the life system is

0.1 that why this procedure do not work in life fast 7500 system.

so, you need see the these specification, and it will work very well no matter which mix you use, in my experience Roche and QIagen work good.

good look

mario

I agree with both the answers above. However, we choose to make our own master mix for greatest affordability. We have been using Syto-9 (Molecular Probes/Life Technologies) together with HotStarTaq (Qiagen) - clearly something that you can't buy.Please also note that if you are doing MS-HRM, it is more forgiving of the platform that is being used than mutation screening protocols.

Qiagen's epitect for rotorgene works quite well. We use it for many different gene targets. I don not know what's going on with other mastermixes, but make sure your bisulfite treatment efficiency is really good

I have used the Qiagen epitect mater mixes on a rotor gene and it works really nicely. Again ensure your bisulfite conversion is good and that the DNA quality is high. For some cpg assays I have had to increase the number of cycles from 45-50 to ensure amplification.

Thank you all for your valuable input. Since our RTpcr machine has an accuracy of just 0.20 ° C, I´ll try the biorad supermix. The qiagen mix might not produce nice results on our machine, since it is designed for the rotorgene or other machines with an accuracy of at least 0.05 ° C. The protocols are also a bit different, as the biorad mix posses a heteroduplex formation step prior to to the HR melting. I just got a free sample of the biorad supermix and will post my experiences. have a nice week! :)

Dear Olivier,

I am currious to hear if it was sucessful as we also use the CFX96 and wonder which mix and assay conditions would be good and sensitive enough for MS HRM.

Dear Edna, 

Yes, I established the HRM method in our lab using the CFX96 along with the Precision Melt Supermix. We already published a paper using this method ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350346/pdf/oncotarget-05-12607.pdf ). I explained it in the Method section. I successfully established MS HRM for around 15 different regions in different gene promoters. 

The first thing I perform is a gradient MSHRM PCR (+- 8°C Tm) with fully methylated and fully unmethylated control DNA and then I choose the Tm with the best discrimination of two peaks, no primer dimers and an Ct value of around 25-30 and then put the product in a gel to check the size. The next step is to  run all my standards ( 0%, 25%, 50%, 75%, 100% methylation) in a very narrow  gradient MSHRM PCR (+-2°C from Tm of the first step) and see at which Tm I get the best linearity of my standards when performing a linear regression. 

Then you are ready to go :) 

I recently used the HRM for detection of SNPs like KRAS mutations. It was no problem to distinguish between wt and two different mutations. 

good luck :)

Dear Olivier,

Which dye did you select when you used CFX96. Because I can not find the Evagreen dye in my BioRad CFX96.

Thanks,

Fuyou

just use the sybr channel, it's the nearly the same wavelength