BSA importance?/ Is Pac 1 a bad "cutter" - Use BSA? (Feb/07/2006 )

Should i use BSA with Pac 1? I have not used it and I get a liner piece on gel picture. But when i transform this (is Alk Phos treated) without ligation rxn in DH10B cells i get colonies on a plate. How is it not cutting? Is BSA impportant?

NEB definitely recommends BSA with Pac I

I often think that phosphatase is a good thing to reduce background, but in my experience it does not eliminate background entirely. I use it as the manufacturer recommends but there are typically still a few transformants that are self-ligated vector...I also think perhaps, even when the enzyme is good and when you cut your linearized vector from a gel, there may be a tiny tiny amount of uncut plasmid remaining in the band too, which will always transform with magnitudes-higher efficiency


I don't know this for a fact, but I have seen that even when you shouldn't get any re-ligated vector on your control plate, there are usually a couple and I think perhaps this is the explanation. it is very hard to get 100% effectiveness of all components. but this is just my theory...

Thanks for the advice...I also found out, on line, that it recommended to be added last. Just thought i would put that out there.