Should I purify digested vector before ligation? - (Jul/31/2006 )
so my freind suggested I try not to cut digested vector out of the gel (maybe UV is the problem) but rather run small portion to see if it's digested, ligate and treat with some RE inside the MCS but not my insert, run negative control and everything will be fine... ...she said i should purify phenol/chloroform after RE digest...but im thinking if im not cutting out of the gel (so no agarose) what am I purifying?? ..what do you think about all that?
phenol/chloroform extraction removes/inactivates protein including REs.
It will work if you dont have small restriction fracgment from MCS together with vector fragment. Otherwise, those will religate back to your original vector.
the only real problem I foresee, is that when you run the band out and extract it, you're getting rid of the uncut vector left behind in the gel. even if you don't see much in the way of bands, there will always be a little left, and this supercoiled plasmid will transform at a much higher efficiency. this would mean that you may be screening lots of colonies, because your negative control (vector-self ligation) plate should give a disproportionate number of colonies
I can see that treating with the RE after the ligation should reduce this problem, but I bet it will not go away entirely
and please make sure to read up on the buffer and reaction specs for each enzyme, if you are only going to purify after the RE (and which RE? the original, or the one that cuts in the MCS?) you need to be careful that there aren't incorrect buffering conditions for any of the components.
thank you everyone very much, i just have one question if im cleaning up from REs then can't i just inactivate it? thanx again a lot....
you also want to change RE buffer to ligase buffer by EtOH precipitation. You dont want BSA and RE precipitate with your DNA. Phenol/Chol removes both.
does it mean that after i ligate and kill the ligation by treating with RE inside the MCS i need to phenol/chloroform again before electroporation???
p/c or column; electroporation requires pretty clean DNA. I suppose you could just drop-dialyze and hope for the best, but your efficiency may be poor
I didn't think the RE 'killed' the ligation? that just prevents undigested vector from the original prep from being transformed? or am I misunderstanding that entirely?
that is excatly what i mean by that... sorry i think i am using the term from the web....