Larissa Levy More times the smear means the high concentration of your DNA and more time this happens when you work with pure culture.

Larissa Levy From their movement on gel, your samples doesn't look like Genomic DNA rather they look like some PCR products, could you please give the details to enable to answer your query?

It may be that in the dilutions and then addition of sample to the pcr mix you are changing the salt concentration of the sample and then the pcr mix.This Might change the running speed of samples so that the highest salt concentration causes the slowest moving band through the gel

Hello Larissa

From the gel pic I feel you need to standardize the DNA extraction procedure. I feel the fungal DNA is getting degraded during the extraction process. Kindly use DNase inhibitors like 2-mercaptoethanol or iodoacetate or sodium dodecyl sulphate.

Thank you.

Ejaj K Pathan thank you! Sorry, I didn't explain correctly. The samples are PCR product with primers AFU5S and AFU5AS (specific for Aspergillus sp.). But, this also happened with ITS1F/ITS2 (universal fungi primers) and ASP5/ASP7 (specific for Aspergillus sp.).

@Larissa Levy, the amplicons obtained with these universal primers shows variation (for ITS, it is between 450-700 no) in sizes depending on the species. Different Aspergillus sp. also shows this variation.

Now the question is you are getting this variation with the same sample but at different concentrations, it can be attributed to presence of more than one species in your sample (i.e. it's not pure preparation).

You can sequence these products to know which are these species, whether same or different?

Ejaj K Pathan should a mixed population of impure template give rise to 2 bands rather than a shift to only one template of a different size being generated ?

Paul Rutland It depends on the primer specificity towards the template and also abundance of particular template.