3 molar insert excess - how imp. is it for the ligation rxn (Jul/12/2007 )
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.
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
Hope this is helpful
For most ligations, even 1:1 ratio would work but for some ligations, we use 1:10 ratio.
1:3 ratio is what I usually end up preparing.