This video gives a helpful demonstration of how to use Snapgene’s program to design primers for Gibson Assembly.įor a simple example of using Gibson assembly, imagine that you want to insert your gene of interest into a vector with a large tag at the N-terminus, but you don’t have the tag already included in the vector you want to use. Both Snapgene and NEB have tools that help you design primers for PCR amplification of fragments to incorporate such regions of homology. NEB recommends an overlap of 15-40 bp, with a primer melting temperature greater than 48℃. The required homology between neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous sequences. Another advantage is that this process makes it easy to generate wild type and mutant constructs at the same time, rather than sequentially. The Gibson assembly process can be used to assemble up to 6 fragments in one step, resulting in scar-free assembly that does not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment. NEB or SGI-DNA), or can be mixed yourself (e.g. The master mix of enzymes can be purchased from a company (e.g. The great part about this mix of enzymes is that they can all work at the same temperature, so the entire reaction takes an hour or less to complete at 50 ☌. After an hour or so, the sample is immediately ready to transform into competent cells. a DNA ligase, to seal the nicks of the annealed and filled-in gaps.an exonuclease, which chews back the 5’ ends of the fragment, generating long overhangs which allows the single stranded regions with homology to anneal.Then, the fragments are incubated together with an enzyme master mix, which contains three different enzymes: To start, you need to have DNA fragments with regions of homology at their ends, which are typically created by PCR. Gibson assembly is well known for allowing easy assembly of multiple linear DNA fragments, but can also be used in basic cloning of an insert into your vector of choice as shown in the figure below. Here at Addgene we added this option to our drop down menu of common cloning options in the deposit process in 2014 because of its gain in popularity. The Gibson assembly technique was first described by Dr. In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to put together DNA fragments.Īs a fun way to start, I highly recommend watching this entertaining video created by our friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “Breakfast at Tiffany’s.” Overview of Gibson assembly They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on. These newer technologies have become more and more common, and for good reason.
Over the past decade, scientists have developed and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning.