PEG ligations summary...

George W Chacko gchacko at magnus.acs.ohio-state.edu
Sat Dec 5 14:40:15 EST 1992


I've tried to summarise the information mailed to me on the use of PEG
in ligations. I'm sorry if I've left anyone out (infact I'm sure I've
lost a couple of messages) but I'm not a very efficient person. Thank
you everyone and my blunt end ligation of a 90bo fragment into
Bluescript II was succesful at 12% PEG after T4 pol blunting a Taq pol
generated PCR product while the control without PEG wasn't.

Regulation of inter- and intramolecular ligation with T4 DNA ligase in
the presence of polyethylene glycol. Nucleic Acids Res 1986;14:7617-31

AB  - Polyethylene glycol (PEG) stimulates ligation with T4 DNA ligase. In
      10% (w/v) PEG 6,000 solutions, only intermolecular ligation is enhanced
      by monovalent cations, while both inter- and intramolecular ligation
      occur without their presence. Similar stimulation was also caused by
      divalent cations or polyamines in the PEG 6,000 solutions. Such
      properties of the ligase could be applied to control the extent of
      inter- and intramolecular ligation. Ligation with cations or polyamines
      in 10% PEG 6,000 solutions was effective for intermolecular ligation.
      Ligation without cations or polyamines in 6.0% to 10% PEG 6,000
      solutions was effective for intramolecular ligation.

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skrypka at aclcb.purdue.edu  
Subject: Re: PEG and blunt end ligations

The reference is "Molecular Cloning: A Laboratory Manual 2nd Ed. Maniatis,
Fritch and Sambrook; pp. 1.70 - 1.71."  Just used it yesterday, worked like 
a charm.  One caveat, if you're using PCR amplified material you'll have to
really blunt end it with something like T4 DNA Polymerase ( Buffers are 
close, except for the obvious lack of ATP for Pol, and Pol can be heat in-
activated at 75 degrees C for 10 minutes ).
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preissj at clvax1.cl.msu.edu>
Subject: RE: PEG and blunt end ligations

I generally don't like PEG in my ligations, and make up my own ligation
buffer to avoid the commercial prep that contains PEG.  It functions as a
volume exclusion thing, increasing the apparent concentration of DNA.
Unfortunately, I find that it tends to sepparate DNA molecules and increase
intramolecular reactions.  In other words, PEG is great for unimolecular 
reactions like self ligations, but poor for bimolecular reactions like
ligation of an insert into a vector.
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preissj at clvax1.cl.msu.edu  
Subject: RE: PEG and blunt end ligations

	I use 2 stocks, first a 10x buffer containing 660 mM Tris-HCl, ph7.6,
50mM DTT, and 50mM MgCl-2.  Then, I use a 10x stock of ATP containing 10mM
ATP.  I keep them separate because ATP breaks down quickly in the presence of
Mg.  BTW, ATP is pH 8 in water.  I store both at -20 dC.  I generally do 
my ligations in 10ul or 20ul volume at 16 dC, overnight.  I try to use about
a 5:1 molar ratio of insert:vector, and between 0.1ug to 1.0ug total DNA per
reaction.  Pretty standard stuff, I think.  Good luck.

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srcksac at chv.lincoln.cri.nz  
Subject: Re: PEG in ligations
The reference I have is actually (I think) for sticky end ligations,
but just in case it can help, or you haven't been sent it already, try
SB Zimmerman & B Harrison. (1985) Macromolecular crowding accelerates
the cohesion of DNA fragments with complementary termini. NARS 13(7)
22241-2249.

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MBWEN at TWNAS886.BITNET  

Ligation buffer supplied by BRL contains PEG and seems to work fine for us.

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rick at geneman.wustl.edu  
Subject: PEG in LB

See Cobianchi and Wilson in Meth. Enz. 152 (p. 109).  Note that what
they refer to as "10x ligase buffer" is actually 5x.
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