Effect of PEG on rxns

Ashok Aiyar ashok at mirage.bioc.cwru.edu
Mon Sep 5 12:47:08 EST 1994


In article 402070b4 at thorin.uthscsa.edu,  HARDIES at THORIN.UTHSCSA.EDU writes:

>
>: Are you sure about this?  Both T4 and E. coli DNA ligases are actually
>: stimulated by PEG.  It is included in many commercial ligase buffers in
>: quite high concentrations. <snip>
>: It may even be leftover primers or nucleotides that you
>: are washing away, but PEG... Naaah.

PEG does stimulate blunt end ligation of large inserts reproducibly in
in my hands.  PEG also seens to have a small inhibitory effect on 
transformation (small but reproducible),

>I first encountered the "PEG is poison rumor" 20 years ago as a
>graduate student.  I tested it by dumping lots of PEG into a control
>reaction and found that the enzyme actually loved the stuff.  The
>rumor gets started repeatedly and always the same way.  If you carry
>over enough sup. after a PEG precipitation, you're just contaminating
>the DNA with the crud you were trying to get rid of in the first
>place.  People often call the sup. the "PEG", since that's were most
>of the PEG ends up.  So the statement "remove the PEG or else ..." is
>often true, but not literally so.   I can imagine one exception. PEG
>has its (usually beneficial) effect by excluding volume and hence
>concentrating the substrates.  As  far as I know, none of the commonly
>used enzymes directly interact with PEG.  But if you get a high enough
>concentration, the volume is so reduced that the DNA can not stay in
>solution; hence it precipitates.  Now if your ligase buffer was
>already full of PEG, and you added more with the DNA, you might
>achieve an inhibition by knocking the DNA out of solution.  This last
>part is conjecture.

The "molecular crowding" effect of PEG seems to be very useful in several
reactions that I do.

Later,
Ashok
--
Ashok Aiyar                              email: ashok at mirage.bioc.cwru.edu 
CWRU Medical School                              telephone: (216) 368-3300 
Department of Biochemistry                             fax: (216) 368-4544





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