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PEG and ligation

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sat Aug 21 07:53:00 EST 1993


In article <1993Aug18.085345.148 at ntet.nctr.fda.gov>, wmelchior at ntet.nctr.fda.gov (Bill Melchior) 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."
> 
>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?
> 

Hi all,
	Having followed this and related discussion for a while, thought
I'd add my 2-cents worth.  We are doing lots of shotgun cloning of 
physically sheared cosmids into blunt ended (Sma1/CIP) treated vectors
m13/pUC and then sequencing.  
	In our hands, the major limit to ligation efficiency seems
to be insert/vector ratio once we have a reasonable amount of vector
there.  As a control for ligation efficiency, because we have to do
a fillin after physically shearing, we use alu digested cosmid and
if all is well, get hundreds of plaques/colonies.  Even so, our cloning
efficiency for the physically sheared/filled-in inserts is maybe only
10% of the alu control.  Thus, we have fooled with conditions to see
what things will help, and concluded that insert/vector ratio is the
most critical factor and there is an optimum (probably due to salt in
the gel purified insert which inhibits ligation at high concentrations).
Anyway, the suggestion is to do several different insert amounts over
a 10-fold range and keep the vector constant at levels that give several
hundred plaques/colonies for the alu control.
	As for ligation conditions......sigh  (lots of witch craft here)
It seems that for us, once insert/vector is optimal, the efficiency
can be improved with PEG and incubation in the refrigerator for from
overnight to 36 hours.  Yes, room temp. incubation works but we've
noticed that upon sequencing the recombinants, there seems to be a
higher level of chewed in vector (i.e. we will see only lots of recomb.
with only one or two C's where there should be three C's from the Sma1
site) with the room temp. incubations.  ALSO, incubation in the frig.
for longer times seems to give many more recombinants than shorter times
in the frig. or room temp. incubations.  Thus, we've settled on overnight
or longer ligations in the frig. and don't do the rt. ligations any more.
	If all you want to do is ligate a single blunt end fragment from
a digest then anything usually works if you have enough insert.  BTW,
for pcr products, we're just doing a gel purification (via phenol extn.
from a low melt) followed by a simple fillin with klenow and the 
4 dNTP's at room temp for 10 minutes and blunt end cloning the product.
The efficiency isn't the greatest, but it works to give enough 
plaques/colonies to get samples for sequencing and to insure no pcr
artifacts.
	Remember, the key step seems to be insert/vector ratio so
do some expts. where you vary the insert amount and keep the vector
constant, and do the alu control to check that your competent cells
are fine and the ligation really is working well.

Best to one and all............bruce
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 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
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