I think that your samples are simply over amplified. and combined with the likelihood that you are amplifying too much dna you are getting minor non specific bands amplifying strongly.

I would measure the od of your dna and use 25ng ( who knows how much dna is in 2.5ul?) and run 30-35 cycles ( 45 is far too many cycles) and include a no template control (water instead of dna) to check for contamination and if you are still getting multiple bands then run a DMSO gradient up to 8%dmso or use a higher annealing temperature.

It may be that your dna contains pcr inhibitors and using less dna will work much better

Dear Airam,

Try to perform serial dilutions before amplification and prefer fewer cycles, as suggested. I usually use 30 cycles with an annealing temperature of 45 degrees and a 3 ul DNA sample (200-300 times diluted, depending on the results of your DNA isolation). Hope it helps. Good luck!

I see that you have used 3 primer pairs. You could use the outermost pair to get an amplimer (dirty) then reamplify 20 cycles of a 1/1000 dilution of the first round pcr using a primer pair internal to the ones used in the first round pcr

verify the quality of your DNA samples and try to make different dilutions