Three-way ligation in two steps? - (Sep/01/2011 )

Could I perform a three-way ligation by mixing two components and ligating, then adding the third component and adding more ligation enzyme?

Perhaps, but why would you want to do that? Why not all three at the same time?

I would recommend not. Remember, the ligase enzyme will keep on ligating, forming concatemers. It will not stop at two fragment. You need the third fragment to 'round' off the reaction and produce your circular plasmid, rather then a linear concatemer.

That's right, but if you do the ligation at low concentrations, and have equimolar amounts of each fragment, a single pot triple ligation works very well. Concatamers form preferentially at high concentrations, where to probability of finding the end of a second fragment is higher than the probability of looping the fragment already formed. This happens around 5-10 ng/ul for normal vector constructs.