Dear All,
I have set up a 5-plex qPCR. The multiplex works fine but the PCR efficiency doesn't look that great. I am throwing in 250ng human genomic DNA and I get Ct in range of 30-35 for all my products. Average Tm for my three sets of primers is 60 Degree. I am currently using a combined annealing and extension of 60 degree with a hot-start enzyme. I realized if I split it into lower annealing maybe around 57 degree and a bit higher extension around 62 degree, it should improvise the overall efficiency.
Any comments? I am using Smartcycler -II
Appreciate your help!
Hi Renu
you can decrease reaction volume to avoid products over use. a PCR of 10 works very well.
what you could do as I do every time when designing primers is to check primers specificity by in silico PCR. if it's for human you can check at the UCSC webpage (http://genome.ucsc.edu) and see also the predicted annealing temps.
fred
The final Ct value tells you nothing about the reaction efficiency of your primers. It just tells you that they amplify late (either low copy number, low efficiency, or a combination).
The only way to measure efficiency is with a standard curve.
You should have already tested the best annealing temperatures for your primers with gradient PCR in a standard thermocycler.
You'll need to think about what exactly you are trying to solve before you start changing the variables. qPCR enzymes are optimized for a very specific temperature range and do not work efficiently outside of those temperatures.
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