multi fragment ligations - (Aug/28/2007 )
This may be a stupid question...
Is it possible to ligate multiple fragments together in one reaction, such as a linear vector and 3 different fragments?
If possible, how difficult is it?
Our basic ligation reaction ligates three parts -- two components and a backbone vector. It works very well, because we have good selection on the final product. The two components come from vectors with antibiotic resistances A and B, and we ligate into a vector backbone with resistance C, then plate out on antibiotic C containing plates. We have zero background from uncut component vectors. We prepare the vector backbone by PCR rather than RE digests, so there is no background of uncut vector there, either. It "always" works (unless we make mistakes, of course). We use Tet, Kan, Chloramphenicol as antibiotics A, B, C, and switch between them as needed for the particular assemby we are doing.
You want equimolar amounts of each component. You want low total concentrations (10-20 ng of vector backbone in a ligation). You want much less ligase than you would think (.05 ul, yes, really). You can do the ligation in 30 minutes at room temperature. We only cut and ligate with four enzymes, EcoRI, PstI, XbaI, and SpeI. You want to grow transformants for 2 hours before plating out rather than the 1 hour recommended in most places.
Multiway ligation is rather simple... the main difficulty is the number of colonies that you need to screen. You will need colony PCR and a multichannel pipette. Thus far the highest multiway I regularly conduct is 5ways (4 inserts and 1 vector). The key part to multiways is to design your fragments so that the restriction site on each fragment end is unique and uncompatible. Thus all the fragments can only ligate to each other in one order and one order only. The next key is the ratio of inserts. You have to accurately quantify how much DNA you have of each fragment and ligate them in a 1:1:1:1:1 mol ratio (assuming your have 4inserts and 1 vector).
A 3 way as Phage434 has mentioned works very well. Although I don't have mutiple markers to conduct my selection, I have a multichannel pipette and colony PCR to compensate. I would say the sucess rate is about 1 in 4, although that would depend on how well you have prepared your vector, and I believe to some degree luck.
4 ways is about 1:25
5 way is about 1:72.
However, I have notice that this is a highly stochastic process. You may have a 4 way ligation and screen the first 50 colonies and not get any hits. But they next 25 colonies gives you 3 colonies. So on principle I will always screen 72 colonies for anything above 3 way ligations. For 5 ways I go to 84 and will reach 168 on occasion. (But with multichannel and a PCR plate that is easy... the major down flaw is loading the PCR on the gel...my multichannel is too wide for my well slots.)
Xgal colour testing is also a very very big help. It is often easier to assemble all your fragments in a blank pBS vector. (Insert ligation disrupts beta galactosidase gene). And later move the combined fragment into your final plasmid (assuming said plasmid has no colour testing phenotype)