There should be no need to change the protocol for your assembly. Just be careful to get all your reaction concentrations correct ( I have spreadsheet calculator if you need it). Make sure to run a negative control with the same amount of vector but no insert.

I recently Gibsoned a 9kb insert into a 5kb vector at a 1:1 molar ratio. Let me know how it goes, the only problem I've ever had with large gibson assemblies was the transformation. I had to use electrocompetent cells instead of chemically competent cells.

Thank you, Christopher. It will be great if you can send me the spreadsheet calculator. I will keep you abreast of my progress.

If I double digest my vector and gel purify the linear vector, is there still the need to do the negative control? My thought is that in that way the background colony with empty vector will be very low.

In a typical ligation, you would expect zero colonies after purifying in the way you described, but the problem is that the exonuclease in a gibson assembly may expose single strands which are compatible enough to anneal.

Almost always I have clean negative controls, but just this last time I saw an amount of false positives which I couldn't ignore. After running a "LALIGN" on 50bp of each end, I saw some pretty decent compatibility, which to me explained how that could have happened.

I do realize negative controls are expensive, but you should probably at least do it your first time. It will help you troubleshoot if you have a problem and may ultimately save time and money. To double your amount of reactions from a Gibson kit, and to justify running negative controls, you can run 10ul reactions instead of the suggested 20ul. I have done this every time and it works. After all, we are worried mostly about concentrations not quantity. You only end up transforming with a maximum of 2ul anyway.

I do this all the time, mostly in 10kb+ vectors. I am still using the home made mix, as described in the original paper:

Enzymatic assembly of DNA molecules up to several hundred kilobases.

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO.

Nat Methods. 2009 May;6(5):343-5. doi: 10.1038/nmeth.1318. Epub 2009 Apr 12.

Perhaps the kit from NEB works even better.

Hi, Koen. Thanks a lot. How long of the overlap sequence do you usually use? In NEB Builder, the shortest overlap seems 15bp, and some online resource suggests 30bp.

Also, for inserting one fragment into a vector, is Gibson assembly a lot better than traditional digestion/ligation clone?

Xiang,

I design 25 nt overlaps sequences into my primers for PCR amplification. For overlap sequences, the emphasis is on achieving a minimum Tm. The Tm must be near or above 50 degrees celsius for overlapping fragments to be able to anneal during the assembly. According to manual for NEB Gibson master mix, the amount of exonuclease is optimized for fragments with up to a 100bp overlap, so there is room for more if needed. The reason to try and keep it near or below 30 is to reduce the risk of secondary structure in the single strand DNA, which would prevent annealing. I analyze my overlapping regions with an online oligo analyzer to confirm the Tm and to look for any bad hairpins.

A ligation is probably only easier if PCR amplification is not needed. For example, the case where the insert and vector are in separate plasmids but have the right restriction sites already.

Hi, may I hijack this thread? :) 

Here is my question: I want to assemble 5 fragments using Gibson, but these do not include the vector, because I want to ligate this assembled piece into different vectors. Basically what I will get is a linear piece. Now, when I do the reaction of assembly, what is the best way to check yield? Will it be clean enough to see a band on a gel?  Or is it better to try PCRing up the assembled piece from the mix? 

I thought about not including the vectors in the assembly, but if you suggest that is the better way to go, please let me know. 

Thank you!