I am trying to get a qPCR protocol off the ground using AB PowerUp sybr green master mix. I tried to use a different kind of ABI machine but got no signal. I later realized this was because (even though I clicked on the tab), the machine never performed a dissociation step. I am now trying to set up a protocol using another machine available to me, a light cycler 96. Does anyone have any experience with optimizing primers and running qPCR on these machines? Thanks!
Hi, SYBR Green reactions should work on any qPCR instrument that can read that wavelength. I have used Roche, ABI, etc, and never optimized between the cyclers. The parameter setup is slightly different for each one, and the analysis is slightly different, but overall they have the same results. They do use different PCR plates, though- be sure to check that! ;)
I'm confused on what you mean by dissociation and the lack of a signal. Do you mean there was no melt curve? You should still have had Ct results. Be sure the instrument is measuring at the correct stage of the PCR- I think ABI is the one that has a magnifying glass that you add in various steps. It's easy to accidentally measure when everything is dissociated instead of when it is associated, and then you have no signal!
I agree with Jennifer's answer. There's no need to do any optimization, since any dye proper for the detection channel of PCR device can generate the signal including in the dissociation stage . It's probable that wrong parameter setting has been made. You should carefully read the device's manual to do the right setting.
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