Hi everyone,

I am trying to amplify the ITS 1 and 4 regions of Aspergillus flavus. My first three lanes, starting from the left, are A. flavus, I then have 2 lanes of C. albicans as a positive control, and then 2 no-template controls for my reaction. You can see my positive control works fine, but for my A. flavus and NTC I have these faint bands below 100bp. Are those just shadows from the loading dye? Or are they product? For reference I am using a 1% agarose gel, 0.5X TBE, and purple loading dye 6X from NEB. I load 2uL of dye and 8uL of PCR product for a total of 10uL per lane.

I don’t see this shadow with my positive control. I know this reaction works because I have run all my yeast isolates with it. I have tried varying annealing temperatures for my molds and they still won’t amplify. They have A260/280 of 1.9 and concentrations of 10ng, which should show up fine on a gel.

Thank you!