Other than assessing for temperature of hairpin structure (for both forward and reverse primers), self-dimer Delta G, and hetero-dimer Delta G, is there anything else to look at before ordering primers?
I have been using applications (OligoAnalyzer; PrimerQuest Tool) provided by IDT to assess for these factors when selecting primers for my gene of interest.
Make sure they are specific to your gene only if you are amplifying from cells. You can try primer--blast. https://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/
Went to the link, entered in my primers, adjusted for amplicon length and organism. I would like some assistance in understanding what I am getting as I believe the result of my search.
Input PCR template: none
Specificity of primers: No target templates were found in selected database: Refseq mRNA (Organism limited to Homo sapiens)
Does this mean that the primer pair I entered is specific to 1 gene, and 1 gene only?
Hi Mikey,
for a good primer GC content should be 50-50% only
and Tm should not be more than 60( better to be 55-60c)
Use Primer Blast for designing your primers. Once you get the list of primers choose one with these specifications and then again go to blast and enter your chosen primer. In the results it should show only your study gene and species name. Then it would work better. Try in this way. All the best.
Hi Mikey
your in silico PCR didn't work. as I can see, you forgot to choose the template, ie genomic or RNA refs.
fred
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