Engineering Customized TALENs Using the Platinum Gate TALEN Kit

Methods Mol Biol. 2016:1338:61-70. doi: 10.1007/978-1-4939-2932-0_6.

Abstract

Among various strategies for constructing customized transcription activator-like effector nucleases (TALENs), the Golden Gate assembly is the most widely used and most characterized method. The principle of Golden Gate assembly involves cycling reactions of digestion and ligation of multiple plasmids in a single tube, resulting in PCR-, fragmentation-, and purification-free concatemerization of DNA-binding repeats. Here, we describe the protocols for Golden Gate assembly-based TALEN construction using the Platinum Gate TALEN Kit, which allows generation of highly active Platinum TALENs.

Keywords: 4-Module assembly; Golden Gate assembly; Modular assembly; Platinum Gate TALEN Kit; Platinum TALEN.

MeSH terms

  • Cloning, Molecular / methods
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics*
  • Endonucleases / biosynthesis
  • Endonucleases / genetics*
  • Gene Targeting / methods
  • Genetic Vectors
  • Plasmids / genetics
  • Protein Engineering / methods*
  • Trans-Activators / chemistry
  • Trans-Activators / genetics*

Substances

  • DNA-Binding Proteins
  • Trans-Activators
  • Endonucleases