Dear all,
I am learning to design the qPCR assays recently. But I just don't know why my negative controls always give positive signal( I have changed my design twice). I checked the formation of primer dimers in different software but all showed my design was good and no primer dimer formed at all.
So I suspect whether it is because my TaqMan probe is too long(26bases)?
Does anyone know will the length of taqman probe affect the specificity of the PCR?
Thanks!
Binbin
Longer probe does not necessarily guarantee better results for sure. We use 15 nt probes in our qPCR run and the outcomes are just fine. It really depends on the sequence of your probe and what your sample is. In our lab, we are developing diagnostic kits using qPCR. The sample is human blood extracted DNA. Even if our gene/primer/probe are specific enough, we still need to do a lot of blast to make sure there are no similar sequences in the human genome.
Hi Binbin
Specificity of a reaction is more depends on the primers than probe. First of all you need to visualize your product on a gel; if it is a single-sharp band with the correct size then you got product already. Second, it come to your probe, length of probe depends very much on the annealing T, you cannot make it longer or shorter for any other reason. There might be some limitations though, e.g. variations inside your amplicon. If you have any, you need to be extra careful.
For the dimmer primer, in most cases softwares can predict it well, however you are always required to screen your product on an agarose gel.
Good luck
Hi,
you can check the PCR product on a gel and in case it is due to non optimal quenching you can consider to go with Zen Probes or other double quenched probes
Thanks
Sven
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