Hello everyone
when i use primers with restriction sites for cloning . so i get double bands when run by gel, while amplifying the same fragment with same primers without restriction sites i get a single amplification.
Probably it happened due to homologous of that gene copy, 1st check the gene sequence similarities in PubMed Blast, you will see which copy of a gene is also PCR, I have this experience much time... Ihteram Ullah
The question to me is not clear enough to be answered. I saw two statements 1) "....other smaller using restriction sites primers after cloning", 2) ".....while amplifying the same fragment with same primers without restriction sites I get a single amplification"
I am not sure about the exact step but I assume that you are getting the problem when you're digesting the fragment. In this case, it is most probable that your fragment contains a restriction site within the frame which is causing the double band. I will suggest to cut your band of interest and go ahead without worrying about the non-specific band.
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