Ligation - dephosphorylation?

[Hinayana Bawagan]

torsten at wst.edvz.sbg.ac.at (KLADE Torsten) writes:


>To all netters!

>Is it absolutely necessary to dephosphorylate the target vector
>when ligating single digested inserts?
>I kept dephosphorylation with additional Phenol-Chloroform extraction, 
>but the efficience of the ligation didn`t seem to improve!
>Any sugestions?

>Thank you
>  Torsten

>----------------------
>Torsten Klade torsten at dgen10.gen.sbg.ac.at
>University of Salzburg

I assume that the following is the sequence following up to 
the ligation step:

1. linearize the vector
2. dephosphorylate the vector (may go directly from the digestion
thus omitting the phenol/CHCl3 extraction and Etol pptn steps)
3. do phenol/CHCl3 and Etol pptn steps and run DNA in a
low melting point agarose gel
4. extract DNA w/ any of the ff: the traditional methods as
described in Maniatis or by geneclean or by Boehringer Mannheim's
agarase 
5. the insert of course should also be gel-purified after
restriction enzyme digestion
6. do the ligation in minimal volume (10 ul) with a large excess
of insert over dephosphorylated vector; also an excess of ligase
wouldn't hurt; have two controls: a) dephosphorylated vector alone
and NO LIGASE to check for carryover of unlinearized vector;
b) dephosphorylated vector alone WITH LIGASE to check for
the efficiency of dephosphorylation; ligation can be carried out
overnight at 12 C, 3-4 hrs at room temp and my labmates got it to
work also at 37 C  1 hr.