Ligation of small insert - (Oct/23/2007 )
I need to ligate an insert of 60 bp to a vector of 4 kb. I have meen performing the ligation at a molar ratio of 1:3 and not obtaining any colonies. I would like a recommendation(s) of vector:insert ratio to use.
I have routinely ligated 90 bp DNA fragments to a 3 kb vector using a 5:1 ratio of insert:vector. 10:1 also works, but ratios higher than that can lead to double inserts.
I use 1:10 ration at least, if I use less, I have lot of negative clones (or if the vector can’t religate, I have no colonies), tfitzwater is right, you can have double insertion, but just sure to check you pick a colony with just one insert.
We use 1:10 ratio for small insert ligations. Although, 1:5 also works. I set up both.