I have changed my extraction method, changed my PCR master mix, changed my water. also I have modified my PCR program but nothing works. and when I got a single band and sent it for sequencing they didn't give me a clear, reliable read.
PS. My primers are ITS1F and ITS2R4.
With lower annealing temperature a partial match between the primer and the template will be stable enough and you would get non-specific amplification. The higher the annealing temperature, the primer will require longer compatible sequence to bind to and as a result your specificity will be higher. Try to increase your annealing temperature.
Some links that you can try for standardization of your PCR..
https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-cycling-considerations.html
https://www.biobool.com/news/181.html
Dr Narmin, In addition to Bhairavi's reply I would also suggest you to optimize the quantity of your template too. Often higher amounts of template generate non-specifics. Next, for clinical samples or samples that are expected to have other organisms too, even if you see a single band in gels, it might contain identical/similar length amplicons from other species/ non specific targets. So you cannot expect a good sequence read from direct sequencing of such amplicons. You have to do a clonal-sequence analyses. best wishes, SD
Annealing temperature must suitable to match primer with DNA template
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