Dear Sharanya

My first impression is that you may have degraded or impure DNA.

What are the conditions of the reaction? conc. starting DNA, type of polymerase, calculated Tm for primers, temperature- and time settings of PCR reaction.

Did you perform another PCR on the same starting DNA as a control for the DNA input? Like a PCR-reaction on a housekeeping gene or other known amplicon?

Good luck

Hi Duco

Thank you so much for the response. 

 as a background for the study I have obtained the cDNA from RNA extracted from tissue. i had diluted the RNA 100 times as the concentration was too high.

the tm for the forward primer was 54.9 deg C and rev was 58.7. and i performed a gradient pcr with the annealing temperatures ranging from 54-60 deg C. 

 And no I didn't perform a control step.

So do you mean to say that my PCR product has degraded or my cDNA itself has degraded?

I would rather think the cDNA is degraded. Please try a well-known PCR on the cDNA to test the quality ot the latter.

D