how about subject to 1.5% agarose gel?

It can be happened. Could be interesting if you try with different combination like 1.5%, 2%, 2.5%  with 10 or 5ul of Ethidium bromide.

Hi, You may try the followings-

1. Reduce the annealing temperature to see if the other band shows up. 

2. Use a 2% TAE-Ethidium Bromide gel. 

All the best wishes!

Thank you!

And yes, I have tired 1.8% and 2.8% gel as well. However, would it help if I tried 1% gel?

Hi Ruwini,

The percentage of gel depends on the size of the PCR product. The higher the size, the lesser the percentage and vice-versa. You may try 1.5 and 2% simultaneously. Again, best wishes!

Is your size standard separating well on your gels?

Are all your results from the same dna sample and if so can you try another sample in case there is a sample mix up or you have a sample with a polymorphism/deletion  on one amplimer under the 3' end of one primer

Is your negative control negative ( no amplification) to rule out contamination from male amplimer taking over the reaction?

what is the OD260/280 ratio of the dna sample?

Are these new primers or have they worked before in your hands?

Hello Paul,

I have been using 100bp ladder and it separates very well in the gel. There were no bands in the negative control ruling out any contaminations. The OD260/280 of the template DNA counted as 1.79 and the primers were newly ordered and therefore we did not get any different result before, but past research proved positive results with the same primer sequence.

Mane yes, we have been using the same template DNA for all the tests and we can try with a new sample.

Thank you for the advice. I really appreciate.