My PCR template was a PCR amplicon amplified from human DNA (nested PCR) which has been diluted 200 times. Taq buffer conc. = 1X, MgCl2 conc. = 2.5 mM, dNTP = 0.2 mM, Taq polymerase = 0.5 U. Four set of primers were used for a single reaction (multiplex PCR).

As shown in the picture, the band with a green arrow was my intended band (960 bp). The smear was stopped exactly at 700 bp and the smear appeared as a 'ladder shape' with discrete bands.

Adjusting the annealing temperature did not solve the smearing problem where every time I managed to make the smear gone, the intended band was also disappeared.

What other parameter that I still can attempt to optimised?

My DNA template is a GC-rich template. Do i have to increase the melting temperature?

Thank you.