impossible single enzyme cut - (Oct/24/2006 )
I'm trying to cut Invitrogen's pcDNA3.2/V5/TOPO plasmid, to linearize prior to stable transfection production. I've used 4 enzymes so far, both GpG meth sensetive & insensitive, but not managed to cut yet. Very odd. The enzymes work fine in other plasmids & for double digests.
Is there a chance that my DNA is supercoiled & I need excess (20 units ?) of enzyme ???
if u r having problems linearising ur plasmid, y bother with digestion, just transfect using any transfection reagent and this should get u a decent % of transfected cells for making stables.