What do your primers amplify? Did you checked for the specificity of your primers using blast search against all relevant databases?

It is hard to tell with the information you providing. Is it a genomic PCR or RT-PCR? For example if it is an RT-PCR it is possible that you have some genomic contamination due to the presence of a small intron. Alternatively, you can also check for the presence of pseudogene, duplication, etc…Contamination by other related species may also occur….

If you cannot figure it out than just send the PCR fragments for sequencing. It is very cheap and you can get back the answer faster than any input back to your question.

Hello,

Even I experienced the same thing that you can see in the above picture while performing one of my experiments.  Two bands clearly state that there are are two separate sized DNA, Now this can mainly be due to contamination that might have happened while performing the set of experiments (as many steps are required to be done), or may that the gene of interest you amplified was not completely and properly done in the PCR, Also its a possibility that the restriction endonuclease might have not completed their job and may their be the possibility of the substrate DNA be present.

These are just the possibilities but you can always troubleshoot by checking steps you performed. Hope this be helpful to you also you can figure out the size of the unexpected band and see if that sized DNA was there at any step of your experiments that might give a clue of what contamination it might be.

thanks for your comments..

hi dear javad

it seems that you work with HWP1 primers for separate  c.albicans complex.

with this primers in some species of c.albicans we get additional band. 

but it needs that we do  more research about this, due to it happens for some species.