Rapid Construction of Recombinant Plasmids by QuickStep-Cloning.
QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was designed specifically to address the major drawbacks of previously reported cloning methods. The fully optimized protocol allows for a seamless integration of a long DNA fragment into any position within a plasmid of choice, in a time-efficient and cost-effective manner, without the need of a tedious DNA gel purification, a restriction digestion, and an enzymatic ligation. QuickStep-Cloning can be completed in less than 6 h, significantly faster than most of the existing cloning methods, while retaining high efficiency.
ChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK.
Pawel
P
Jajesniak
ChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK. t.wong@sheffield.ac.uk.
Tuck Seng
TS
Wong
D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
D004274:DNA, Recombinant
Ligation-independent cloning; Megaprimer PCR; Metabolic engineering; Plasmid construction; Protein engineering; Recombinant DNA; Synthetic biology