Integration of PCR Fragments at Any Specific Site within Cloning Vectors without the Use of Restriction Enzymes and DNA Ligase | Zendy

Martin Geiser | Zendy; Régis Cèbe | Zendy; Delia Drewello | Zendy; Rita Schmitz | Zendy

Open Access

Integration of PCR Fragments at Any Specific Site within Cloning Vectors without the Use of Restriction Enzymes and DNA Ligase

Author(s) -

Martin Geiser,

Régis Cèbe,

Delia Drewello,

Rita Schmitz

Publication year - 2001

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/01311st05

Subject(s) - plasmid , restriction enzyme , dna ligase , restriction site , cloning (programming) , biology , multiple cloning site , restriction digest , cloning vector , in vitro recombination , dna , genetics , molecular cloning , computational biology , mutagenesis , recombinant dna , microbiology and biotechnology , gene , vector (molecular biology) , computer science , mutation , complementary dna , programming language

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 is 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™ Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel 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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