Multiway Ligations; sharing the art - Three way ligation and above (Sep/02/2006 )
I was wondering if there are any multiway DNA ligaters here? I would like to compare and trade protocols, tips, observations and short cuts.
To start the ball rolling…
The largest multiway ligations I do is 5 way. Each fragment is within the range of 1kb to 3kb. All sticky ends are made incompatible by restriction sites hooked on by amplification primers. I keep my fragment ratio at 1:1:1:1:1 mol ratio, though lately I have been experimenting with success of 1:1 ratios of fragments and a 2:1 ratio between fragment and vector. I do dephos my vector but do not kill digest my ligation mix, (cause I find this restriction digest too time consuming to be bothered with). I use Xgal/IPTG colour testing to remove colonies with unligated pBSsript vector. I use colony PCR and my trust multichannel pipette to screen large number of colonies.
I have some questions too… Is it possible to perform multiway ligations with vectors within the 10kb to 30kb range? Is so could you please share your wisdom. I have little luck here.
Has anybody played with their ligation buffer formulation? I use the standard NEB stuff, but is there anything better?
Are ligation kits that much better? They cost blood. My supervisor is tight on cash and time so I can’t go out and buy a kit (there are so many out there, any recommended) and play with it.
Has anybody attempted larger mutiway ligations? 7 8, 10(?) Is so, do you have any advice to maintain ligation efficiency? I think I am experiencing problems with ligation products 10kb in size. But I am not sure, as I haven't done a proper experiment.
And finally thank you.
We routinely do 3-way ligations as part of "standard assembly" of biological parts (http://parts.mit.edu). The two inserts come from vectors with antibiotic resistance A and B. We cut with pairs of enzymes, heat kill, but do not purify the product. We make "construction backbone" by PCR of a plasmid backbone carrying antibiotic resistance C. We cut this PCR product with a pair of enzymes, and clean up the short ends (improves efficiiency). We mix equimolar amounts, ligate, transform on C plates, and go. The backbones from the input inserts have the wrong resistance. There is no plasmid with resistance C unless it is formed by the triple ligation. We use antibiotics tetracycline, kanamycin, and chloramphenicol, and rotate between them -- you can always choose a C which is different from A and B. All plasmids are also Amp resistant, which makes preparing master plates and distributions easier. The enzymes we use are always the same -- EcoRI, XbaI, SpeI, and PstI.
The result of these assemblies can be used as input to a subsequent round of assemblies. For assembling N components, you need log2 N stages.
Well I guess that is about all the responce I will get.
Even so I want to say thank to Phage434 for sharing the idea of selection marker rotation. It will certainly be added to my cloning repertoire.
And before I finish; adding 5% PEG 4000, increases the rate of ligation by crowding the DNA molecules together. (Found this tidibit when searching the archieve of the forum)
EDIT: Thank you DJG for that piece of information. I'll give it a try.
If you want to increse the ligation efficency, after CIP reaction you could incubate your sample (Vector, fragment ligation buffer) for 5'min at 65°C. After that, add the enzime, ATP and proceed with the ligation reaction. This allows vector to be more relaxed in order to facilitate the fragment insertion!
Just a comment on ligation buffers: we use the 2X rapid ligation buffer that comes with promega's TA cloning vector kits (pGEM T-Easy, for example) - and nicely enough, they provide you with the complete recepie for the buffer (just check the online protocol for the kit), we make it according to that and I have the impression that ligations work more efficiently than with other commercial 10X ligation buffers.