b'Cloning large and complex genesCloning large or complex genes can be challenging. Many standard cloning methods can introduce additional DNA or scars that interfere with gene function. However, there are several established methods for the assembly of larger genes from multiple fragments. These include Gibson Assembly cloning and type IIs assembly, also known as Golden Gate assembly.GeneArt Gibson Assembly HiFi overviewGibson Assembly cloning is a highly efficient way to assemble up to 15 DNA fragmentsdsDNA fragments with overlapping endssimultaneously with vector sizes of 100 kb or more. In the first step of assembly with3 5Invitrogen GeneArt Gibson Assembly EX Cloning Kits, a 3 DNA exonuclease digests5 3fragment ends to allow annealing of homologous segments. DNA polymerase fills inAdd Gibson Assembly the gaps in the second step, and DNA ligase seals the nicks to generate a covalentlyEX Master Mix Abound DNA backbone. The DNA construct can then be used to transform chemically3 ends chewed backcompetent or electrocompetent cells. 3 55 3In type IIs assembly, adaptors containing type IIs restriction sites are added to the donor DNA to facilitate transfer of multiple fragments into one or more recipient vectors. TypeFragments annealIIs assembly kits can be used to assemble up to eight fragments in any order to create a3 5construct with a maximum recommended size of 13 kb, including the vector.5 3Learn more about seamless cloning by visiting our DNA cloning tips page. Add Gibson Assembly EX Master Mix BDNA polymerase closes gaps(3 extension)5 33 5DNA ligase seals nicks5 33 5Overview of annealing. With a GeneArt Gibson Assembly EX Cloning Kit, you can reliably assemble up to 15 inserts in a two-step reaction.Find out more at thermofisher.com/gibsonassembly Contents 9'