can I just ethanol precipitate Klenow? - without deactivating it? (Nov/08/2006 )
i am self ligating. after Klenow i forgot to deactivate it and just ethanol precipitated. i want to ligate after that. is it enough to get rid of Klenow? can it interfere then somehow? thank you always!
ethanol precipitation doesn't get rid of proteins; it gets rid of salts and allows you to concentrate
to remove the protein, there are options such as gel purification or column purification, but phenol/chloroform will be the fastest, I think
The Klenow subunit has now cursed your DNA. Unless it is removed it will degrade 3'overhangs produced by restriction enzymes like ClaI... and fill in your 5'overhangs with As... (well it probably add one)... Doom, Doom.... (it is getting late....)
Options have already been suggested by amikins. Also agrees with Amikins, though would like to add the phenol/chloroform is also the cheapest option.
thanx a lot! i see your concern. however im not going to digest it again right away, im electroporating it into E-coli and then miniprep. do you think it will still be effective?
Was the Klenow used for a partial fill in for a "blunt end to blunt end" ligation?
If no overhanges are involved, then you are okay for ligation and subsequent electroporation.