PCR amplification with high fidelity enzyme
Posted 18 February 2013 - 12:44 PM
I have a rather stupid question I have a genuine confusion about. I'm trying to amplify a gene from a plasmid that is 9.6 kb in size (the whole plasmid) and my gene to amplify is 4.2 kb. I'm using a high fidelity polymerase (NEB Phusion). What should be the size of product expected (i.e is it a minimum of 4.2 kb and higher depending on when the polymerase falls off the template ?) I'm getting to see a smear with a minimum 4 kb band and higher but the really bright band is 6 kb. Is this spurious ?
Posted 18 February 2013 - 12:58 PM
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.