A low concentration of a primer than the other one in high concentration or even in volume often exhibits good melt curve appearances.
What could be the reasons behind this? Is this dependent on level of expressions (Low / High) or copy numbers of GOIs or what?
Looking forward for some detailed explanation.
Hi,
Higher concentration of the reverse primer compared to the forward primer, result in more of the strand complementary to the probes being amplified and allow more signal to be generated.
You can refer to this ref. oi: 10.1128/JCM.40.4.1571-1572.2002 J. Clin. Microbiol. April 2002 vol. 40 no. 4 1571-1572
Good luck in your research
check carefully the primer dimerization and/or self dimerization... if you didn't check before. maybe the "excess" of one of the primers allows self-dimers or other less-specific annealing in your reaction.
this is a simplified tool to perform primer sequence analysis.
http://crn2m.univ-mrs.fr/AmplifX?lang=en
also, you should not exclude some interfering contamination in your primer aliquot (usually contamination effects are reduced by diluting reagents...)
in both cases, these are actual triggers of low-quality curves...
mlk
Just to expand a little on MLK's response, in addition to checking the primer sequences with online tools like Primer3 blasted against a mispriming database and Vector NTI to verify/screen the sequences you got from Primer3 (should be done prior to ordering primers if you're designing your own), I'd also suggest running each primer through a PCR thermal gradient and spectroscopy via Nanodrop. You can then rest assured your working with pure and specific primers, and you'll also have optimum annealing temps. Makes trouble shooting downstream much easier when you know the primer sets are solid...hope this helps
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