Hello Pranay,

Biotinylated amplicons should migrate with slightly decreased electrophoretic mobility but the difference will be obvious on 1.5% gel? I am not really sure. Have you tried 2% or 3% gel? Why not do a southern blot to confirm biotin labeling?

Adding strep-AP directly in PCR reaction? I am not sure about that but I guess if you do that and electrophorese the mix, you may notice a band shift compared to non-biotinylated PCR product. It's just a wild thought. I don't know if it would work though.

Hi Pranay

As Syed mentioned there is probability of electrophoresis shift of biotinylated primer. Since you are asking about primer, its certainly difficult to see on agarose gel. 

I suggest use some Biotin chemistry, to try binding it with Avidin/Streptavidin and run on an PAGE gel. Four Avidin (64KDa) molecules binds Biotin with good affinity. So, in presence of Biotin you can see protein band at 64*4=256KDa region on an non denaturing PAGE. 

So, you can proceed with your guess, rather than doing an agarose electrophoresis, try running it on PAGE

Hi

I notice that neither agarose (1.5, 2, 3 % gels) electrophoresis nor PAGE (10%) were able to resolve the issue as the molecular weight difference between unlabeled and Biotin labeled is too less to be resolved on an agarose or PAGE [244 D which is less than 1 bp difference (650 D)].

Dot blot assay helped me to find out labelling efficiency!!

Thanks for advices Syed and Arun