A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products

Sci Rep. 2019 Apr 23;9(1):6417. doi: 10.1038/s41598-019-42868-6.

Abstract

An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes. Here, I describe the development of three vectors for TA cloning and blunt-end cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR cloning protocol with non-digested circular vectors and PCR products. This direct PCR cloning protocol yielded colony-formation rates and cloning efficiencies that are comparable with those obtained by conventional PCR cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene ( https://www.addgene.org/ ).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Cloning, Molecular / methods*
  • DNA Primers / chemistry
  • DNA Primers / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Engineering / methods*
  • Humans
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Polymerase Chain Reaction / methods*

Substances

  • Bacterial Proteins
  • CcdB protein, Plasmid F
  • DNA Primers