Instead of using normal agarose gel electrophoresis, use 40% -50% native PAGE to separate close fragments.

Hello Tushar,

Separating such closely migrating fragments by electrophoresis sounds next to impossible. However, it should be possible to enrich one of them by PCR using adequately designed primers.

If possible I'd recommend to restriction digest the fragment you don't need using an additional enzyme. Chopping it down to 2 smaller fragments you'll leave the other available for clean gel purification.

Alternatively PCR amplify the fragment you want to transfect and DpnI digest the reaction.

Hope it helps

Francesco