You do not show a size standard so it is hard to say but it could be primer dimer because you have too much primer in the pcr mix . If you use less primer 1/4 or 1/8 of what you are currently using or use a hotstart Taq the dimer should disappear and you will also get more pcr product. Without a size standard it is also hard to say that the pcr band is 400 bp as well

You should always include a DNA ladder in your Agarose gel. It makes it easy to determine size of PCR products and makes it impossible to confuse orientation of where your samples are in the gel. You could analyze another lane which is a negative control. Everything same concentration, little more water, but no template DNA. Put that in the thermal cycler, as well, and you see what a primer dimer looks like.

Also the color resolution looks kind of faint. Did you know that you can cast agarose gels with Ethidium bromide? They solidify with the dye.

In RFLP longer incubation times might degrade DNA or nonspecific digestion of DNA. Secondly DNA quality and isolation should be good isolation yields good template. lastly the buffers you were using should be made from nuclease free water. and the DNA ladder is a must to know whether the bands are of right size or not.

• Agree with Paul
• I note also that your actual putative band is much weaker than conglomerate at bottom suggesting that the PCR reaction is of low efficiency; also implying the bottom material is primer dimer

Thank you Paul adron beera laurence

I reduced the amount of primers but with the same resault and the smear seems to be above the primer dimers

P.s: I stopped using ladder for pcr after i was sure of primers and bands because ladders are expensive and very hard to import to here .

This is an image

Ok so ideally use a hot start enzyme. If this is not possible then with a band of 400 and a digest then the dimer should be about 100bp. This means that any digest with one cut site will produce a band you can see. eg if the cut is 100 plus 300 then you may lose the 100 band but the 300 band proves the presence of a cut site even if you cannot see the small band. You may just need to run the gel at a lower voltage and for longer so the dimer is well separated from the amplimer to see the one or 2 larger sized bands. Meanwhile you could try measuring the OD of all of your dna samples as some are amplifying well and others are over amplifying. Some of your bands are quite weak.If you have a smaller toothed comb to make smaller wells that might make the bands easier to read