mlsulliv at wisc.edu
Thu Mar 23 10:03:37 EST 2006
You don't need to dephosphorylate if the overhangs from your
restriction digestions are not complimentary (which in all liklyhood
is the case if you are using two different enzymes-- an exception for
enzymes that produce 3' overhangs might be something like PstI and
NsiI which have different recognition sites, but the same overhang).
Incompatible overhangs have little, if any, potential to ligate to
each other so there's no need to dephosphorylate.
Usually, you only need to dephosporylate your vector if the cut ends
of your vector are compatible with each other (e.g. cut with the same
enzymes or with different enzymes that produce the same overhang- and
it doesnt' matter if the overhang is 3' or 5') or if the ends are blunt.
On Mar 23, 2006, at 2:21 AM, newsnet customer wrote:
> I have a vector that has been digested with 2 different restriction
> enzymes to remove a DNA fragment - I expect 2 bands on a gel.
> Anyway, both cuts produce 3' overhangs. I was wondering that since
> these sites are unique I could do directional cloning with a new
> DNA fragment. My question is that, do i need to dephosphorylate the
> ends of the cut Vector? I thought since the ends are 3' overhangs
> there is no way of them religating.. or am i wrong?
> Sharp Tool
> Methods mailing list
> Methods at net.bio.net
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
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