ligation problem - (Nov/29/2006 )

hi all,
i was trying to do a 3 piece ligation, and for some reason it wasn't succesful,
i'm doing it for the first time, so i don't have much expirience,
i thought about doing it in a different way but i don't know if it's possible, so i'll ask your opinion:
i thought about taking the 2 inserts (2.6kb and 1.5kb) and ligate it together without the vector,
and then i want to try to insert the "big" insert (made of the 2 inserts) into my vector,
what do u think? is it possible to do it this way? will it work?
thanks for your opinion,
Anat

My feeling of your proposal is that the inserts will continue ligating to each other, forming some very large concentamers.

Not stopping at a simple
AB heterodimer but will continue to something like 'BAABBAABBAABBAA'

I think it best to add the vector directly into the mix to stop the reaction from progressing to super size concentamers.

The important thing to remember about multiway ligation is the vector:insert ratio.

The molar ratio (NOT MASS (ng) ratio) between insertA and insertB must be 1:1. Extreme deviation from that will kill the multiway ligation as the excess insert species will ligates to all other component preventing, circulisation.

The vector:inserts molar ratio can be 1:1, and I have also tried ratio of 1:3... I have not noticed any significant difference between the two ratios.

thanks,
i really haven't thought about that problem,
i hope i will be able to make the ligation the regular way..

in this forum i read before that there are some other people who tired to ligation 3 pieces of fragment but they failed. so it better to perform ligation step by step

Theoretically, it should work. But practically, it could be tedious, involving many trials (it may even work the first time).

Conservatively, I would suggest ligating one piece into the vector first, do transformation, get new plasmid, cut it and repeat with the second fragment. For this, you should design the restriction ends of your first fragment to be able to ligate to both ends of a cut vector. It just involves an extra step in designing the restriction ends of the fragment.

You can also put in the vector, the first and the second fragments in the ligation mixture and prey for a miracle. It did work for me a couple of times, but it really is like winning a lottery.

Also, you could start with as much of each fragment as you can purify from a gel, ligate together, then run the resulting mess on another gel. You ought to be able to see enough of the ligated product to cut it out of the gel and re-purify (this is why you want to start with A LOT of the two input fragments).
From there, it should be easy.

Alternatively... ligate one piece to cut vector, purify the resulting molecule, and ligate the second piece (which will 'insert' into the vector+first piece molecule). This might work better.

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Member of The Science Advisory Board since February 2006
The Science Advisory Board

umm...... I regularly do 5 way ligations. So from where I stand 3-ways taken for granted that it will work. 6-ways is admitedly bit of a challenge.

I would call doing 2 way ligations tedious. (Not to mention impractical for the work I am doing.)

So Amigon, despite the words of caution you are hearing, 3 way ligations is okay.

Just make sure your design plan is has all inserts ligation with directional ends, the primers are designed properly (there is sufficient bp surrounding the restriction site for the enzyme to work), make lots of product, gel purify all your DNA fragments, use good ligase and ligase buffer, get your insert:insert ratios right. Don't over dephosphorylate your vector, is it easier to dephos lots of vector (reduces shocastic error and pipetting error).

I tend to agree with Perneseblue. 3-way ligations can be quite friendly when each fragment has different incompatable sticky ends.