I am using SYBR Green qPCR mastermix in Bio-Rad CFX-96 qPCR machine. For a 25 microlitre reaction of normal PCR, I use 0.75 miclolitres of 20 picomolar concentration, each of forward and reverse primers and it gives satisfactory bands in agarose gel. Should I use the same concentration and amount of primers for Real Time PCR when the reaction volume is 20 microlitres per reaction?
After the QPCR check your product on gel. If you see primer dimer, or non-specific fragments then you could lover the primer concentration. 0.4uM final concentration is standard. Hope that helps.
After the QPCR check your product on gel. If you see primer dimer, or non-specific fragments then you could lover the primer concentration. 0.4uM final concentration is standard. Hope that helps.
Thank you very much @Petre Ianakiev Sir. will try that next time I perform the qPCR experiment.
We use 10ng primer for qPCR. The machine we use is of applied biosystems. But, I don't think machine make matters.
I use 2µl of an equal mix of F and R primers, each at 5µM, in a 20µl reaction.
This is using Roche Sybr on a Lightcyler 480.
I've never seen primer dimer when i ran the products.
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