5 replies to this topic

[baccilus]

How to do HRM curve analyses with SYBR Green Dye using ABI 7500 RT-PC? I have only done absolute quantitation using commercial kits from ABI up till now. In my new lab, I need to do High Resolution Melting Curve analysis to look for Gene mutations. I have SYBR Green in the lab and an ABI 7500 RT-PCR. Please suggest how I can accomplish this. I would be really thankful. If possible, please point me towards a manual or protocol.

[baccilus]

Can anyone please have a look at my problem. I have the ABI Syber Green Mastermix. But I have no idea of how to conduct an HRM analysis with it. Since ABI has it's own HRM kit, they don't provide a protocol for performing HRM with the SYBERGreen Master mix.

[Trof]

SYBR Green is not a good dye for HRM. It's not saturating the strands and when denaturing the molecules jump to different places so the fluorescence isn't proportional to the melting status of DNA.

You have to buy a special HRM dye or a kit.

[baccilus]

Trof, on 14 September 2010 - 04:11 AM, said:

SYBR Green is not a good dye for HRM. It's not saturating the strands and when denaturing the molecules jump to different places so the fluorescence isn't proportional to the melting status of DNA.

You have to buy a special HRM dye or a kit.

Thanks for the reply. I am using the SyberGreen Mastermix out of necessity and not out of choice since I don't have the ABI HRM kit. Moreover I will also have to buy the HRM software from ABI separately. I know the SYBERGreen master mix is not the best choice but AFAIK, it can still be used. I would be really thankful if you can point me towards some good guides on this specific subject.
Thank you in advance.

[Trof]

There is an article with several references about SYBR Green in HRM on the page I linked before.

[baccilus]

Trof, on 14 September 2010 - 06:49 AM, said:

There is an article with several references about SYBR Green in HRM on the page I linked before.

Thank you very much for this. I don't know how I missed this.