PEG and ligation

Anne-Francoise J Lamblin nimbus at molbio.cbs.umn.edu
Sat Aug 14 16:24:58 EST 1993


PEG 8000 at 5%(w/v) in the ligase buffer has been shown to improve the ligation
efficiency of blunt end DNAs.
The reference is a 1986 BRL Focus "optimizing DNA ligations for transformation"
Vol. 8 #1 (winter issue).
For the ones who cannot get this issue , the buffer was the following:
5x ligase buffer:
250mM Tris-Cl pH 7.6
50mM MgCl2
25% (w/v) PEG8000
5mMATP  
5mM DTT.

Aliquot and freeze at -20.
The conditions recommended for ligations of blunt end insert into vector DNA 
were the following:

1x buffer
1 unit or BRL T4 ligase ( BMB will do the same job)
vector/insert molar ratio =3 ( up to you to change it )
4 hours at room temp (23-26C) 
Dilute 3 to 5 fold before adding DNA to competent cells.

This study also shows that 4 hours of ligation (blunt ends) at room temp gives
gives the same result as 23 hr at 4c! (2x 105 transformants/microg DNA).
This buffer is also compatible with ligation of inserts with sticky ends  or addition of linkers to insert DNAS at 14C.

They did not mention anything about sonic ligations but I can tell you it works mainly for the sticky ends not for the blunt ends.

Anne-Francoise Lamblin
University of Minnesota
nimbus at molbio.cbs.umn.edu



More information about the Methods mailing list