The length of the homologous ends depends on the number of fragments being assembled and their length (see below). PCR primers need to be designed to amplify your fragment of interest and include 20 or more base pairs of homology to the vector or adjacent fragments in your cloned product. ► Learn to simulate Gibson Assembly in SnapGene Insert Preparation You have the option to prepare your vector by linearizing your plasmid with restriction enzymes or by using Reverse PCR. Insert fragments are always prepared by PCR. To perform Gibson assembly, you will need to prepare one or more inserts and your vector. This article refers to all these reactions generically as Gibson Assembly. Second-generation assembly reactions such as HiFi Assembly and Gibson Assembly Ultra improve on these limitations. The classic Gibson Assembly reaction can result in errors at fragment junctions and does not work with single-stranded fragments or short fragments. The reaction is designed to occur at 50☌, with all enzymes selected for their stability and activity at this temperature. T5 exonuclease chews back the 5’ end of double-stranded DNA to expose engineered overlaps. Polymerase begins filling in at the overhangs preventing excessive enzyme chewing. When stable compatible overhangs anneal, ligase completes the fusion of the DNA fragments. Once ligated, the DNA is protected from the exonuclease. These enzymes work together to fuse overlapping DNA fragments.
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