In-Fusion cloning

14/07/10

Home
USER Protocol
ATMT Protocol
Fast gDNA Protocol
Chemical comp. E.coli
Electro comp. E.coli
Electro comp. A.t
Xi-cloning
In-Fusion cloning
Crossing Fusarium
S. M. Extraction
Macrocondia

 

 In-Fusion cloning is a commercially available kit from ClonTech. It is supplied either as a liquid enzyme or as a dried-down cloning mix in 0.2 ml PCR tubes. The technique is dependent on the action of a 3' single strand exonuclease that digest the ends of DNA thereby generating single stranded overhangs, that are able to anneal to compatible ends of other DNA fragments.

I tested the kit back in 2006 for its ability to perform four-fragment cloning, and initially obtained a cloning efficiency of 10 %, however later attempts to reproduce these results did not yield any correct colonies, possibly due to batch variations in the dried-down enzyme mix provided by the supplier.

Several laboratories have had good results with the technique for fusion of multiple PCR amplicons in a single cloning reaction. I have used the technique for construction of vectors for targeted gene replacement in fungi.

 

Advantages:

The technique allows for the directional cloning of PCR amplicons into unique restriction enzyme sites of any vector.

 

Please check the manufactures home page for further information on this technique (www.clontech.com). 

Home | USER Protocol | ATMT Protocol | Fast gDNA Protocol | Chemical comp. E.coli | Electro comp. E.coli | Electro comp. A.t | Xi-cloning | In-Fusion cloning | Crossing Fusarium | S. M.  Extraction | Macrocondia

Dette sted blev sidst opdateret 14. July 2010