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A Guide to Gibson Assembly from the University of Cambridge, UK; Gibson Assembly Primer Design Tool; Gibson Assembly Site Directed Mutagenesis Primer Design Tool; Chemical Transformation of Gibson Assembly Constructs; Perkel, Jeffrey M. (January 2014). "Seamlessly rewriting the lab cloning manual". Tech News. BioTechniques. 56 (1): 12– 14.
The overall success of OE-PCR based DNA assemblies relies on several factors, being the most relevant ones the instrinsic features of the DNA sequence to assemble, the sequence and length of the overlapping overhangs, the design of outer primers for the final amplification and the conditions of the PCR reaction.
The Gibson assembly method is a relatively straightforward DNA assembly method, requiring only a few additional reagents: the 5' T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase. The DNA fragments to be assembled are synthesised to have overlapping 5' and 3' ends in the order that they are to be assembled in.
Gibson assembly is a quick cloning method that uses three primary enzymes; 5' exonuclease, polymerase and ligase. [15] The exonuclease digests the 5' end of DNA fragments leaving a 3' overhang. [15] If there is significant homology (20-40 bp) on each end of the DNA insert, it can anneal with a complementary backbone. [15]
The 4R/2M assembly method was designed to combine parts (BioBrick Assembly Standard 10 or Silver Standard) within existing plasmids (i.e. without PCR or subcloning). The plasmids are reacted in vivo with sequence-specific DNA methyltransferases, so that each is modified and protected from one of two restriction endonucleases that are later used ...
Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for the assembly of large DNA oligonucleotides from shorter fragments. The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process.
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Primer Premier is a bioinformatics software used for various PCR applications. It supports the design of degenerate primers for amplifying a related set of nucleotide sequences for the detection of common traits amongst organisms, as well as to determine heredity. [1] The software also designs tagged and nested primers for multiplex PCR ...