Plasmid digestion and gel purification using HindIII - (Oct/25/2013 )
I'm trying to digest a plasmid using Apa, Klenow and HindIII enzymes to ligate an oligo. I don't have any problems with Apa and Klenow, but when a digest using HindIII and then purifier the agarosa's gel with a DNA purification kit I can't see any band, but I know there is DNA. I use the DNA purification kit for klenow too, and it works ok.
Does anyone have idea why I can't see the band in the agarosa's gel after HindIII's digestion and purification?
How much DNA are you loading? How big is the fragment?
I´ve tried with diferent volumes and I have the same problem every time. The fragment is a 5,3 pb plasmid from which I get two fragments after the HindIII digestion, a 3,2 pb and a 2,1 pb fragment. I can´t see those fragments in the electrophoresis but I know for sure that I have some DNA there being that I decide to continue with my experiments using a tiny and weak band and the experiments turned out well. So, why I can´t see the bands in the electrophoresis??
Ok, those are biggish fragments, and should certainly be visible on a gel IF you are loading enough DNA - when I said "how much?" I was meaning in terms of mg or ug, volume is irrelevant unless the concentration is also supplied.
You would probably need to load a minimum of about 100 ng to see those bands. Depending on how you normally make a gel, you might want to try not including EtBr or other staining dye and do this after running, this decreases the background and increases the specific staining, as well as eliminating EtBr shadow.