Role of PEG in ligation - (May/30/2006 )
hi all
what is the precise role of PEG in ligation...i take it that it is useful in blunt end ligation, but how...also, what if sticky end ligation is carried out in the presence of PEG, will there beany effect, good or bad...
viv
hi
you ay have a look here :
http://www.bio.net/bionet/mm/methods/1993-August/007112.html
Briefly, "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."
There is a PEG based quick liagtion kit from NEB (i think)which is great in ligation but u could not use it for electroporation of the PEG mixed DNA. U have to heat shock the cells.
thanks scolix, i used the fermentas instaclone kit, where neither electroporation nor heat shock are required. they have their own solutions to treat the bacteria and the rec plasmid, and the bacteria get transformed. transformation with this kit has worked well, but i had a problem with the basic t/a cloning and i had posted earlier a query or two. i lost patience and have ordered primers with restriction sites included, and am waiting their delivery.
the reason i had asked was that i have used two systems, pgem t easy of promega, and instaclone of fermentas. the latter used peg, and the former did not. the point was that both are t/a cloning kits, and peg is supposed to help in blunt end ligation. with a single "a" and "t" hanging out from the insert and vector respectively, i wondered if this would be considered a "blunt" end cloning...
thanks fred, for providing the url
-viv