Hello Chai community,
I have a unique protocol that I’m trying to develop and it requires a piece of equipment that doesn’t currently exist.
I routinely need to do a preparative amplification of a template with an unknown concentration and without over-amplifying. Unfortunately, given the ranges of unknown input, optimal amplification can require anywhere from 12-38 cycles. In order to determine the correct number of cycles, we currently take a few aliquots of the reaction and run them for a staggered number of cycles, and, after running a gel to determine optimal amplification, apply that to a large scale reaction with the rest of the material. I need enough material at the end to do an in vitro transcription so it’s typically a 1.5 mL reaction divided up into aliquots.
I’m trying to find a way to spike a dye into one of the aliquots, and use a qPCR to monitor fluorescence as the samples cycle. What I need is a way to program a qPCR to, rather than run for a fixed number of cycles, monitor the reference sample and use a pre-defined fluorescence value as a trigger to finish the run and go to 4 degrees forever.
So a qPCR is actually significantly more sophisticated than the type of “smart cycler” that I actually need, and while undoubtedly not an existing feature of the software, I was wondering if this was a plausible use that one could program for this device.
Thank you for any insights or suggestions you can provide.
Also, sorry to the Chai folks for also submitting this as a support ticket, but I figured the community might be a better place to have this discussion.