PEG and ligation
Michael G. Kurilla
mgk2r at Virginia.EDU
Mon Aug 16 08:45:17 EST 1993
nimbus at molbio.cbs.umn.edu writes:
> 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
> 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.
In a followup article (Focus vol. 8, no. 3, page 13), BRL
claims that overnight ligation at 14C produces 4 fold more
colonies after transformation relatice to the RT for 4 hours
> 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.
Incidentally, you can purchase the BRL ligation buffer without
buying their ligase. Just ask for it when you order. It's pretty
cheap and saves time and effort involved with making all the
separate reagents and worrying about buffer components when
cloning efficiency drops off.
mgk2r at virginia.edu
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