How do you optimize your PCR? Does one of the bands have the correct size? Try different annealing temperatures (possibly times as well depending on the length of your expected product), primer concentrations and check your primers for specificity and secondary structures

you can also try to variate MgCl2 concentration and template quantity. if you don't have a thermocycler with gradient temperature, try several conditions, varing one parameter at once (template, magnesium, primers) with all the possible combinations. if it doesn't work, you could design new primers

Hi.....

Its look like either the concentration of the primer your using is high thus surplus amount of primer is binding to non target sites

Solutions: 1. Reduce the primer concentration, If after reducing primer concentration also its not worked out then try option 2

2. Reduce the Mgcl2 (Use different concentration of mgcl2)

3.If above two are not working then try to optimize template concentration keeping the least concentration of primer constant

4. Nothing is working then try to optimize annealing temperature of the primer by keeping gradient pcr

6. Still not working then primer designing is problem design new primers according to your need

I'm agree with Mr. More, and may be you can check the genome or optimation process of Reverse transcription

Regard

did you blast your primers?

Huma, it depends of your experiment. In your particular target gene, are your "double bands" looking as in the attached picture? 

It might be that one of the 3 main haplotypes of this gene is the cause and not the PCR optimization in general. The probability to get "double" bands in your so many different DNA samples remains high if your primers are encompassing the polymorphic regions VKORC1 381 and 9041 and you find wild type. Well optimized PCR reaction can simultaneously identify these nucleotide polymorphisms (SNPs) so probably you have checked on wild type samples. The probability of having wt samples is as high as 63% based on published data in Caucasians.

Hi Huma:

I think if you increase your annealing temperature you might get a single band or may be your primers are nonspecific and they are binding to 2 different sites of genome, which you can resolve by designing the primer having specificity consisting of up to 25 nucleotides.

Very right ..this problem is very common during standardization of amplification of any template, but as discussed above the annealing temperature and primers designing and their concentrations are generally responsible for these type of problems.