Testing newly designed primers prior to real time - (May/16/2007 )
I have a question about testing new primers for real time PCR.
I designed several sets of primers for my assay based on homologous genes in other organisms, using Primer 3. The genome for the organism I am workign on has not been sequenced. I have done this in the past and it has worked well.
This time, there were very few choices of primer/probe sets because the sequences available for homologous genes are short. I only really had 2 options. One set did not give a product that was visible on a gel. The other set gave a product, but its relatively faint compared to the positive control (28S rRNA).
My question is, does the faintness of the band matter? Is any product visible on a gel enough proof that the primers bind, and I should just order the probe?
you could test the primers with real time pcr using sybr green, if you are not sure about efficiency, primer dimers or unspecific amplification.
Thanks Ned. I hadn't thought of that as I haven't used Sybr green. Makes sense