I ran an experimental ddPCR with 2 different annealing temperatures (60 and 65C) and 2 different probe conc.. At 60C and 200nMprobe the separation b/w negative and positive droplets looks good (the amplitude is around 7000 for positive droplets) but at 65C and 300nM probe the amplitude is around 3500. The melting temp. for the FP and RP are 55/56 and for probe it is 60C.
Does annealing temperature makes this difference or probe conc. making this effect?
How could it be explained to understand this pattern?
Thanks
Annealing temperature has a big effect on the fluorescence amplitude of the positive droplets and the bright to dim ratio.
Briefly:
The higher your annealing temperature, the higher your specificity, but the lower your efficiency.
A lower specificity might generate a better efficiency, but if you go too low the specificity might go down.
To optimize an assay, you can run a gradient on the annealing temperature.
For primer-probe conditions, I would just advise the standard ones. (I would not spend time optimizing those, except if you go for radial or amplitude multiplexing)
You can find more info in the ddPCR applications guide p43 (including an illustration): https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf
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