Standard Cloning Methods

Three approaches for assembling DNA fragments encoding TALE repeat arrays using standard restriction digestion and ligation cloning methods have been described in the published literature:

REAL is a method for assembling DNA encoding TAL effector repeat arrays of any length developed by the Joung lab (Sander et al., Nat Biotechnol. 2011). With REAL, a user assembles together DNA fragments encoding single TAL repeat domains using standard restriction digest and ligation reactions to create a final assembled array. TAL effector repeat arrays assembled by REAL are identical in DNA sequence to those made by the Joung lab FLASH method.

"REAL-Fast" is a faster version of REAL recently developed by the Joung lab. REAL-Fast follows the same assembly protocol as REAL but utilizes plasmids encoding pre-assembled TAL repeats (originally developed for practicing the FLASH method) rather than single TAL repeats (Reyon et al., Curr Protoc Mol Biol. 2012). TAL effector repeat arrays assembled by REAL-Fast (like those made by the REAL method) are identical in DNA sequence to those made by the Joung lab FLASH method.

B. Zhang and colleagues have also described an assembly method that uses standard restrictiondigests and ligations (Huang et al., Nat Biotechnol. 2011). This strategy also involves hierarchical assembly of DNA fragments encoding single TALE repeat domains.

Protocols

For REAL assembly

For REAL-Fast assembly

For assembly method of Zhang and colleagues

Reagents

For REAL assembly:

Set of 28 plasmids encoding single TAL repeat domains from Addgene

TALEN expression vectors from Addgene

The two sets of plasmids above are also available as a combined kit from Addgene

For REAL-Fast assembly:

Archive of 376 plasmids encoding pre-assembled TAL repeats (available by request from the Joung lab)

TALEN expression vectors from Addgene

For the method of Zhang and colleagues:

Plasmids available by request from the authors

Software

ZiFiT Targeter for REAL assembly (publicly available web-based software that provides graphical assembly guides)

ZiFiT Targeter for REAL-Fast assembly (publicly available web-based software that provides graphical assembly guides)

   

Home | Assembly Platforms | Newsgroup | Links | Contact