Reaction volume for ligation - (Nov/24/2007 )
Hi,
How does one decide when to use a large or small reaction volume for ligation reactions? My protocols have seen as little as 10 ul to ~5 ml final reaction volume.
thx for any thoughts.
Most people use low reaction volume.
People use large volume only when intramolecular ligation (self-ligation) is the intended product.
How does one decide when to use a large or small reaction volume for ligation reactions? My protocols have seen as little as 10 ul to ~5 ml final reaction volume.
thx for any thoughts.
I usually use 20ul. What matters I think is the amount of products you want to ligate. The ratios of nucleotides and stuff I guess
Since we only use 1 ul for transformation, anything much above 10 ul is just a waste of chemistry, unless we are running a gel for analyzing the results.
we typically use 20ul but I have also used 10ul for ligations. All of them work. You have to choose a protocol for yourself and be comfortable with it.
For molecular biology and ligation of DNA fragments, I don't think you need more than 20uL. I usually use 5 for transformation and run the rest on a gel, and even that is a lot.
Most important is the ratio of your fragments to be ligated. Too many pieces (i.e. using too high of a volume/concentration) tends to inhibit the reaction.