What are the chances of intra- vs. inter-molecular ligation?
If the DNA is very concentrated, inter-molecular ligation is more likely. If the DNA is dilute in solution, then intra-molecular ligations are more likely. Therefore, the following is sound advice: • If you desire to circularize a single linear molecule, the ligation should be performed at low DNA concentration. • If you desire to combine two different DNA molecules by ligation, the concentration of each should be high – on the order of 1 micromolar. The optimal molar ratio of “insert” to “vector” is generally taken to be about 2:1. Remembrance of things past… Do you remember in your old chemistry class, how a careful distinction was made between the concept of “concentration” and “activity?” You may recall that square brackets [DNA] were used to indicate concentration in an equation, whereas round brackets (DNA) were used to indicate activity. Here’s where that little pearl of wisdom will finally become useful! It is possible for us to change the solution characteristics so that (DN
