wmelchior at ntet.nctr.fda.gov writes:
> Regarding the use of ligation accelerators like PEG or hexamminecobalt,
> one thing about them that has always worried me is statements like the
> following from Sambrook et al (page 1.70 of the second edition of
> Molecular Cloning):
>> "They alter the distribution of ligation products. Intramolecular ligation
> is suppressed, and the ligation products are created exclusively by
> intermolecular joining events. Thus ... all the DNA products are linear
> multimers."
>The idea behind PEG is that it is volume excluder, so that your
DNA and enzyme are concentrated into a smaller volume than you
set up. Intermolecular ligation is dependent on the
concentration of the DNA. Intramolecular ligation is
concentration independent in terms of the rate. Obviously if
you use more your output would be greater.
For most ligations you are ligating two pieces together, so the
first step is inter (and accelerated by PEG) the second is now
intra (and won't be affected by PEG). It's the first you want
to speed up. The Focus article in BRL is good to review
because they used transformation as the readout (not evaluation
of ligation products on a gel). Thus they optimized PEG and
the dilution after ligation for getting the most clones which
is what most of us want.
> I assume this can't literally be true, or the transformation efficiency of
> the ligation products would be very low, but I'd be interested in comments
> on this aspect. For instance, if I want to put a blunted insert of a few
> hundred bp into a plasmid of 4-5,000 bp, would I be better of using PEG or
> HAC, or not?
>IMHO, you are better off using PEG for all blunt ligations.
> ________________________________________________________________________________
> Views expressed are not necessarily those of NCTR, its sponsoring agencies,
> or the United States government.
>> Bill Melchior ||
> National Center for Toxicological Research || ALL statements
> Jefferson, AR 72079 || in this
> (501) 543-7206 || signature box
> || are false.
>WMELCHIOR at NTET.NCTR.FDA.GOV ||
Michael Kurilla
mgk2r at virginia.edu
