# 3 molar insert excess - how imp. is it for the ligation rxn (Jul/12/2007 )

Hi.
Previously what I have been doing for ligation rxns was
1> cutting out my vector and insert with common RE
2> gel extracting it
3> running it on a gel and viewing the intesity of the vector and insert bands
4> quatifying the vector and insert based on the intensity
5> setting up ligation rxn with 1ul vector and 3ul and 1ul insert>
6> It worked most of the time

But now everything is at hault with the ligation. Now I realize that there is a calculation for the ratio and can anyone please let me know the importance of this and how it effects my ligation. I know that too much insert forms concadamers, that limits the rxn. I want to know how much is too much.

As a part of trouble shooting I have sought advice from different individual bloggers, and I am thankful for helping me hone in to few crucial steps like the lesser inincubation time for SAP and inactivating it and others.
any other advice would be appreciated.

-pras45-

In part it depends on weather you use sticky ends or blunt ends. For sticky ends a 1:3 (vector:insert) ratio is usually sufficient for successful ligaton. Blunt ends are a little more difficult to ligate, so I use at least 1:10 and go up from there if necessary. I always want to use more insert than vector just to push the reaction in favor of my desired construct.

As far as calculating it goes, I estimate the concentration from the gel. The band intensities should be enough to estimate concentration (ng/uL) as long as you know the concentration of your ladder bands. I've found using a spec to be too imprecise for measuring the concentration of fragments for cloning. But just knowing the conc is not enough, you need to account for the size to get a proper molar ratio.

I use the following formula: ((ng vector x kb insert)/kb vector) x (moles insert/mol vector) = ng insert

So I choose the ratio I want to use, and the amount of vector I want to start w/ (usually 10-20ng) and calculate how much insert to use for my ligation from that