Amplification of 2nd PCR product
 

I am using a "two step PCR" method in order to generate point mutation
in plasmid DNA. Recently I have encountered into a strange problem.
By trying to amplify the 2nd PCR product with the two exterior primers
I get an unrecognized fragment, which differs in length of the 2nd PCR
product!
The reason I have tried to amplify the 2nd PCR product was a low yield
of the expected fragment, probably due to a formation of by-product
identical in length to the by-product received from the
"amplification" attempt.

What is the cause to this phenomenon?

Thanks

Gil Leichner
The institute of Lipid research
Sheba Medical Center
Israel