why dephosphoration of vector is not recommended? - (Apr/28/2006 )

Hi all,
I read from some protocol saying that when doing plasmid construction, the dephosphoration of vector is not recommended in that it will "increase the frequency of rearranged or deleted clones",
this I don't understand, can someone help explain a little to me (I dephosphorated all the vectors I used)?
Thank you!!

-HLH-

I think that it depends on the vector.
But in general, if you dephosphorat your vector then the ligation isn’t preformed well.
So you actually have a plasmid with an insert that is held together only by hydrogen bonds.
I can’t see how a blunt end will work at all.

-molgen-

QUOTE (HLH @ Apr 29 2006, 12:35 AM)

Dephosphorylation is done simply to reduce the plasmid religating, which drops your number of false positives.

Ifyou're constructing a plasmid, do you do it one fragment at a time, or everything together?

-swanny-

For blunt ligation, one would dephosphorylate the vector to avoid religation. But I have not heard that "it will inc. frequency of rearranged or deleted clones".

-scolix-