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Double Ligation - (Oct/10/2005 )

Did any of you ever try and succed with a double ligation?

I have a vector and two inserts that have to be put together.

-vector----|BAMHI|***|BAMHI|---INSERT 1---|HYP99I|***|HYP99I|---INSERT 2---|NOT1|***|NOT1|--vector

* - ligation places

the end product should be a circular plasmid


I have had this work, but it is very inefficient. I let the ligation go longer than normal, I guess I don't know why, but I thought it would take longer.


We do this routinely in a suicide vector for creating chromosomal deletions. We screen transformants by colony PCR for correctly formed plasmids. Making and detecting these clones has not been an issue, nor has mating them into the recipient strain and selecting meridiploid strains. Getting them to cross out and leave the mutant behind has been a bit tough on occasion...


I have did some, and I feel it is case by case. That time I wanted to fuse my protein with 3 different affinity tags, so I cutted three vectors in the same way with 2 RE. The two inserts (promoter and cDNA) were also treated with suitable restriction enzymes, just like your plan. I did the 3 double ligation reaction at the same time, and the result is: one construct gave me 100% correct colony (10/10), one was (1/20), the third one was 0/(30++).

Try your luck tongue.gif


ouch... I think I'll give it a try, maybe I'll get lucky

Did you use the t4 dna ligase? And what temp? I usualy do my ligations at 16C o/n


Try to first ligat two of your fragments, then add the third one. It should help.


QUOTE (clairebr @ Oct 12 2005, 01:25 PM)
Try to first ligat two of your fragments, then add the third one. It should help.

If I do it that way, I might get a big concatomer someplace, but if I force clone the two inserts into the vector at once, it might close up with just the two inserts I want.


It shouldn't with the overhangs you have.


there is no way I'd do it this way unless I had to.

It is much easier if you break it down into sub-cloning steps. Instead of spending four weeks of "clone, screen, and pray," you will get it done in less than two weeks and have some useful stuff sub-cloned.

Don't make Molecular Biology any more complicated than it needs to be, I say. Baby Steps and Keep it simple, Stupid!