I want to do to genotyping of the a sample and designed the probes with many softwares but all are giving me probes with tm below 59 deg. as per qPCR guidelines I tried making primers at 4-5 deg below probe Tm. During optimization my reaction is giving dimers and a little no product intensity is visible. I am using combined annealing and extension step. Can anybody advice me the change in protocol to optimize low Tm probes.
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Richard Bruce McQualter
If you have access to a gradient PCR machine, run your reaction with annealing temperatures ranging from 5 degrees below, to 10 degrees above your calculated Tm. You will probably find that you are using too low an annealing temperature. Also your template may be too concentrated.
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