Truthfully, I don't think it matter too much anymore. Primer 3 was what I used previously.

What is important is which primers you choose. There are some guidelines on primer design for different assays. For example, for Taqman, product is usually 100bp, and the probe, I just pick whatever will fit in the middle.

Also, there are guidelines for temperatures based on GC content of the primers and the product.

Hi Halley,

primer3 is a free tool but it's just an aligner, not giving the exact characteristics of the target and all probes and primers.

Beside all rules for primer designing, I prefer to use Oligo7 (https://www.oligo.net) and test after all primers on in silico PCR tools made from UCSC (http://genome.ucsc.edu) or NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) to test correct targeting.

fred

Thanks for your answers, Frederic Lepretre Shen-An Hwang.

Shen-An really? I design my probes with high specificity, manually, then next, I used software and platforms like Primer3, PrimerBlast, and others and looked for a pair that ranges the probe site, so as you said I think it will work, cause I tested the design at AlleleID software and it showed me that primers that I had already draw would work well and gave me only one probe and it matched with one probe designed for me manually. Do you think that I could use this pair of primers designed to work well and a probe with high specificity in the middle? Shen-An Hwang

Hi Halley K. C. Soares , so my targets were fairly routine and I never had too much issues with my primers and probe selection. From what you described, the primer and probe set should work.

If, when you run the PCR and it doesn't work, you might have to optimize your protocol.