PCR amplification with high fidelity enzyme - (Feb/18/2013 )
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 ?
depends where on the plasmid the primer binding sites are. if they are very close to the insert the size will be around 4.2 kb, if they are more distant, the product size can be bigger.