Journal
BIOTECHNIQUES
Volume 31, Issue 1, Pages 88-+Publisher
FUTURE SCI LTD
DOI: 10.2144/01311st05
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Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange (TM) Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multi-parallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.
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