TITLE: Gibson Assembly Cloning
AUTHORS: Addgene the nonprofit plasmid repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes Gibson Assembly cloning (</span><a href="http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Nat Methods 2009;6(5):343-5</span></a><span>). To see the full abstract and additional resources, please visit the </span><a href="https://www.addgene.org/protocols/gibson-assembly/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;:;">.</span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a></div><div style = "text-align :; float : ;"><img style = "" src = "https://media.addgene.org/data/easy-thumbnails/filer_public/cms/filer_public/15/c4/15c45cf9-3d03-4f61-93e9-c39159f6916e/gibson_assembly_overview_1.jpg__700x351_q85_crop_subsampling-2_upscale.png" /></div></div>

[STEPS]
?. Design your plasmid and order primers (see figure below).When designing your plasmid, think about what DNA segments you will need to join to create your final plasmid. Adjacent segments should have identical sequences on the ends (sequences A and B in the figures). These identical sequences can be created via PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals to the target sequence.
One strategy is to order primers that are 60 bp long, with 30 bp matching the end of the adjacent fragment and 30 bp annealing to the target sequence.
Avoid strong secondary structures in the homology region. Hairpins in this region can significantly reduce the efficiency of two homologous ends annealing.
?. Generate DNA segments by PCR (see figure below).
?. Run the PCR product on an agarose gel to check for size and yield. If there are significant amounts of undesired product, gel purify DNA segments. Otherwise PCR purification or even the raw PCR mix can work fine in an assembly if you want to save time.
?. Combine segments in Gibson Assembly Reaction.The Gibson Cloning Master Mix consists of three different enzymes within a single buffer. Each enzyme has a specific and unique function for the reaction:T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other.Phusion DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments.Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment.Phusion DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments.Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment.Incubate the mix for  at  or follow manufacturer's instructions. You can purchase master mix from a company (e.g. NEB or SGI-DNA), or make your own (ex: Miller Lab Protocol).
Note, yields will be best when the the DNA fragments are present in equimolar concentrations.
50 °C
?. Transform the DNA into bacteria and screen for the correct plasmid product by restriction digest.
?. Sequence the important regions of your final plasmid, particularly the seams between the assembled parts.