I'm having trouble obtaining a well-defined band of the COI gene in mollusk (Aulacomya atra) adductor muscle samples, despite using 35 cycles and TBT-par. Other mitochondrial and nuclear genes amplify smoothly using the same samples, such as the mitochondrial 16S gene and the nuclear 18S and 28S genes. What recommendations would you provide to optimize the PCR and achieve a clearer band?
you are using 100nM dna only for this primer set and that seems a lot; As other pcrs are working on this dna the most likely reasons for failure are old primers, pcr inhibitors in the dna and too high annealing temperature, You could try increasing the Mg by 25% and running an annealing temperature gradient on 59ng/ul of the dna or just reguce the annealing temperature by 3c if you do not have a gradient pcr machine. If the primers are inherited from another researcher and not new then they may be depurinating if made up in water and not TE. Double the amount of primer to see if this makes a difference to amplification efficiency and consider ordering new primers. Check the exact sequence of this gene in this species to check that there is not a single base sequence change under your primers lowering the annealing temperature,
My guess is that the primers are the most likely reason for the pcr not looking perfect
You can use another type of DNA polymerase for example Phusion polymerase which is highly specific and has a low elongation time with the presence of ssO7D, or even KOD polymerase which has proofreading ability, that will decrease the error rate that you can face
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