Large Fragment Gel Separation - (Jan/20/2004 )
This question will probably have a real easy answer, but I figure I'd ask anyway. So, I'me cloning a 3.3 kb fragment into a 3.5 kb TOPO vector. Cutting out my insert will obviously leave me with two fragments only barely distinguishable on a 0.8% gel. I need to purify my insert for further cloning. Should I be running this on a higher concentration of agarose and running the samples (much) longer for better seperation. Or is there something I'm just completely overlooking? Thanks for any help!
Do both. High conc. gel and longer run.
you should check whether it is possible to use another enzyme that cuts in your vector and not in your insert. As a result, your 3.5 kb vector band can be reduced in two (or more) bands of smaller sizes. There's bound to be an enzyme that does this. It would make things alot easier.