The caveats you could experience with your approach may be akin to re-inventing the wheel in a way... in which case you might risk spending more money than you would otherwise going with the proprietary products already marketed. However, it's not unheard of to discover something brilliant using your empirical approach. But remember, things like Eva Green exist out there as well... (minor groove binders in some cases, that may prove to be more efficacious than SYBR Green I). Companies exist for a reason ... and not all of them divulge their secrets ... for reasons of reasonable (non-greedy) profit in some cases. There are "fluorescence stabilizers" that are added (which are proprietary) to qPCR master mixes which may help things like SYBR Green I do its job. What those "stabilizers" actually are ... may still elude every hopeful empiricist out there - in theory. Just adding "A" to "B" may be a 3-year long quest is what I am saying. But, I am a staunch believer in empiricism, nonetheless.

Jack, thanks a lot for your helpful warning! I have been using SYBR Green I premix for five years but you know during the current funding situation, I am thinking of saving every penny. It seems that I will still have to use commercial premix.

BTW, Jack, it is a nice job, Jack M. Gallup. "Difficult Templates and Inhibitors of PCR" PCR Troubleshooting and Optimization: The Essential Guide. I would recommend your publication for the PCR beginner.

Dear Jinhua Wu, you are too kind... Thank you. Money has been a real hassle here too (ever since 2005)...but, before that too. My heroes are Bustin, Vandesompele, Hellemans, Pfaffl, Nolan, Hands, and Kubista. Without them, (and crucially, Suzanne Kennedy of BioTechniques Forum and MO BIO Labs), I would have never written my chapter. Meeting some of them in person was a great inspiration. It was like meeting a great classical piano player and not knowing what to say that would seem original. I have since resigned myself to rubbing the belly of the Buddha - with a smile - for good fortune. And it has worked. ;]

Hi

You can find very cheap but good mastermixes at Solis

https://www.sbd.ee/

maybe you can save time and money.

Francesc, thank you very much for the helpful info. It seems really cost-effective. I will check if they have agent vendors in US or not. Happy Holiday!

Can I ask where you bought your SYBR separately? I'm having a similar dilemma but cannot find a source.

Thanks in advance! :)

I didn't try to buy them separately since my project doesn't need many QPCR any more.