double digestion and dephosphorylation - (Feb/23/2006 )
i digested my vector with AscI (3' protruding end) and SmaI (blunt end). wht procedure should i need to follow for dephosphorylation??????????????
can any one pleaseeeeee reply me???/
Are you cloning into this sticky->blunt end vector? If so, I don't think dephosphorylation is totally necessary. The sticky end shouldn't ligate readily by itself with the blunt end, but make sure you properly purify your digested DNA.
Check out dephos enzymes at NEB they have some of the more popular enzymes and have procedures included: search dephosphorylation
CIP from NEB gives good results.
I have had pretty decent luck with their new one, antarctic phosphatase....and it is new enough you may get a cheap deal on the price still?
the sticky end shouldn't ligate readily by itself with the blunt end, but it can ligate with other vector (same vector) , like 2 same vector will ligate to the same end that is sticky to sticky and blunt to blunt. so i want to avoid these situation .
from moleculat cloning (sambrook et al) i found the same procedure of dephosphorylation for 3'protruding end and blunt end, so i am going to follow it.
i use CIAP for dephosphorylation.
well for generla ligation procedures, it's for avoid the vector sef religation or concatemerization of vectors that excess of insert is added.
To be honnest, i've often get religated vector, but none concatemers...