ligation question - three-point ligation (Mar/22/2009 )

I was thinking about trying a three-point ligation with a non-directional cloning approach. The details include insert "A" cut with XhoI/BamHI, insert "B" cut with BamHI/XhoI and "the vector" cut with XhoI only.
I realize that most likely the vector will close up on itself upon ligation (even though I plan on using a phosphatase prior to ligation). My real question is whether there is a decent chance that I can favor the insertion of A+B into the vector, at all to be able to detect it. Has anyone had any success with something like this or is it practically impossible?

Could I try and ligate the inserts (A and B) together first and then add vector to the ligation mix?

Thanks for the soundboard.

Hey

I have never tried it but would go for ligating A and B first, precipitate them or column purify and then add dephosphorylated vector. For some reason triple ligations never work in my hand. But I know of people who do it and get it.

Best,
TC

I have not attempted a 3-way ligation in this format before.

Is orientation important?
Can you do X-gal colour test?
Can you do colony PCR to make the initial screen? (With a multichannel pipette)

This reaction will be difficult, but I think you can isolate your clone, provided you are able to screen 100-200 colonies.

yoda1311 on Mar 21 2009, 09:46 PM said:

If you do, you should gel purify this ligation mix and extract the band which correspond to ligation of A+B fragment. It would be helpful if you could differentiate this band from A+A and B+B bands. If you can differentiate the A+B band from the A+A and B+B fragment, this step would greatly improve your success.

However do note, that dues to the nature of your fragments, formation of long concatemere is also very possible. so you might be losing a fair bit of DNA.

I've never tried this myself but have heard of some rare instances of it working but more times than not it doesn't seem to work out. Also, you are going to wind up screening many, many colonies unless you are very lucky.

Just wondering, maybe someone here could answer, is there any way to screen the initial ligation mixture and specifically amplify the A+B ligation product in PCR before ligating into the vector? I'm not sure if this would work and would like input from others but if you take the the first ligation reaction which will contain a mixture of products, purify, use it in a PCR with a "A" specific forward and a "B" specific reverse, would you be able to specifically amplify the A+B product you are hoping to isolate? Can anyone tell me if this might work?

We do this type of three-way ligation routinely without much difficulty to create plasmids for allelic replacement. The keys are to have a good vector prep that has been correctly dephosphorylated and gel purified, and to gel purify the insert fragments after the double digestion. Also, having a good ligation prep and a good preparation of competent cells.

rkay447 on Mar 22 2009, 06:38 AM said:

This is why colony PCR and multichannel pipette is used. Screening 96 colonies is just 8 repeats of a 12 channel pipette. 100 or even 500 individual colonies can screened without much work. The average 3-way ligation only need a screen about 48 colonies to isolate 2-3 colonies carrying the desired plasmid. You just need to work with good quality DNA.

rkay447 on Mar 22 2009, 06:38 AM said:

Yes, your idea does work. This idea is used primarily to build genes from synthetic oligonucleotide.

In normal multiway ligation, the ends of the fragments are incompatible. So a colony PCR screen is sufficient to find the desired plasmid.

We routinely ligate three DNA fragments, but avoid the religation of the vector by double digestion of the vector with different restriction enzymes. With good DNA and antibiotic selection against the vectors used for the inserted fragments, we get near 100% correct assemblies. I think I'm doing badly if I have to screen four, let alone 500. Pick one, or serially dilute the transformation and pick the last well that grows, miniprep, sequence verify, and go.

I agree with the points phage434 makes. The reason why we don't use a doubly digested vector is because the suicide plasmid into which we need to clone these fragments has very few restriction sites available. If we had a better engineered plasmid, we too would use a doubly digested vector, and if such a vector is available to you, you should do it this way too, and avoid the somewhat tricky dephosphorylation step.

I have the opportunity to clone into the vector by way of a directional, double digest. However, the sites are kind of close together (18bp) so I have no readout to allow me to isolate fully cut versus partially cut vector (vector is 7.5kb). Is there any approach that can highly favor the fully cut form, or conversely any technique that can allow me to detect the fully cut versus partially cut vector fragments?

You can make a vector having an insert between the two sites you want to cut at, and then the digestion produces two easily distinguishable fragments which can be gel purified. If you don't want to do that, you can PCR with outward directed fragments around the vector, and add whatever restriction sites you'd like to the (now linear) vector DNA. Cut with the RE's and go. If you want to get fancy, use 5' biotin primers on both ends, and remove uncut or partially cut vector PCR products with streptavidin beads.