Inhibition of BamHI - (Jan/22/2007 )
Is there any obvious thing that could inhibate BamHI activity?
I am trying to prep a backbone digested with HindIII and BamHI, and the restriction profile looks kind of OK, except that I am supposed to get a fragment out, fragment that I can see, but that seems very weak to me... And as I can not clone anything in the backbone afterwards, I was wondering...
BamHI is quite sensitive to salts. Try to first cut the plasmid with only BamHI and optimize (maybe you need an extra ethanol precipitation and washing), when you are happy with it, do the double digestion/add HindII/ or precipitate your DNA and continue with HindIII.
If u start with a higher DNA conc. of the plasmid, you will c a brighter fragment ( its proprotionate to the starting DNA conc.).
You wrote that u do c a faint fragment band, Where did the vector run according to the ladder, did it run as if its digested or not? The ladder might give a better picture if the plasmid is digested or not.
bamHI has star activity so be careful of digestion time
yeh I know, I tried to be careful... I guess I could have also asked if there is any condition that can enhance BamHI star activity... I use conditions (amount of enzyme, digest time...) that worked before, but maybe if there is "something" different in my solutions (water for instance???) they could become wrong???