I have a question - is it possible that my digested PCR product appear as not clear band due to protein content in reaction?
I digested plasmid, and it appeared as quite nice bands on gel. Then I use this digested fragment of plasmid in fusion PCR with 2 fragment of genomic DNA (both of them were too extracted from agarose gel by commercial kit) and PCR product again digested. After this second digestion I run gel electrophoresis and I had smeary band in a fusion PCR product size and smear around the area where gDNA fragment should be found. May the protein from "digestion mix" (or protein/DNA ratio) affect product or template?
In original article authors recommended phenol:chloroform extraction and ethanol precipitation before every gel extraction, but we avoided this in attempt to minimalize losses.