Hi Tuan,

Gibson cloning is useful if destination vector does not have a proper restriction enzymes sites, and also useful for inserting long fragments of gene of interest.

When you clone a larger fragment, look out for Tm and G/C in the overlap regions.

You can also go to vendor's website and read more about, as well ask for assistance in trouble shooting.

Hope this helps and good luck with your cloning!

Ramja

One weakness of Gibson assembly: repeated DNA sequence wont make effective overlapping flanking ends for each DNA fragment. However, you can overcome this with better design or using another cloning technique.

Though not explicitly listed the advantages and disadvantages the following chart gives you some ideas on what assembly to use under what circumstances:

https://www.neb.com/tools-and-resources/selection-charts/synthetic-biology-dna-assembly-selection-chart

In addition NEB and Addgene has great resources on this methods:

https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly

https://www.addgene.org/plasmid-reference/cloning-choice/#Isothermal

I'd agree with the previous answers that it depends on your problem. I would say that, for a "vanilla" cloning problem (e.g. 1 kb gene into a pET vector), I would now always use Gibson. Especially with the up to date kits, the efficiency is very high (much more so than ligation cloning). An effective approach with standard vectors is to digest the vector with a restriction enzyme, and then design the gBlock to overlap this.

One issue with gBlocks is that you really have to get multiple clones and sequence at least two. Gene fragments are essentially PCR products: they are an ensemble of DNA with the right size, which has been sequenced to check that the ensemble has the desired sequence. However, the individual DNA unit that ends up in your vector may still have an error. IDT (the only company that I have used) have a low error rate (maybe 80-90% of 1 kb fragments clone in with the correct sequence) - other companies might not be quite the same quality.

It's worth considering the option of a full gene synthesis/cloning package, depending on your problem. The best suppliers are now offering prices that are not much more than a gBlock anyway; and the cloning cost is similar to the reagents that you would need for cloning (if it all works first time). I'd say that unless you are doing fairly high throughput cloning, or need a super-fast turnaround, the economics now weigh in favour of getting the cloning done for you, once you factor your time in.

I agree with all the answers mentioned above. I am adding some more comments. Gibson assembly is first of all a modular method which if you master it you don't need to deal with different restriction enzymes. In here you don't need any restriction enzyme but mastering the primer design, overhangs, etc.

If you have a short fragment like when you want to build a library it may be problematic because the exonuclease will eat all the fragment and make it difficult to repolymerize. But of course there are ways to overcome these kind of problems.

Goldengate is also a very useful method that have been shown very high efficiency. Goldengate could be even more modular in a way that you need to design your primers somehow to be complementary with the other PCR fragments. You can also do subcloning when using goldengate when having more than 3-4 fragments to assemble.

In overall these new methods are modular and standardized and you can assemble multiple fragments always with more or less the same protocol.