Protocols
- Enzyme-Free Cloning (Daniel Tillett and Brett A. Neilan, Nucleic Acids Research)
Describes a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation–hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This ‘enzyme-free cloning’ procedure is highly efficient, and is not constrained by the need for the presence of suitable restriction enzyme sites within the plasmid vector. The avoidance of post-amplification enzymatic procedures makes the technique rapid and reliable, avoiding the need for multiple sub-cloning steps.
http://nar.oxfordjournals.org/cgi/content/full/27/...
Added: Sun Oct 25 2009, Hits: 1933, Reviews: 0 Write review - Ligase Independent Cloning (Bitesize Bio)
The protocol deals with vector preparation, T4 DNA polymerase treatment, annealing and transformation
http://bitesizebio.com/2008/01/17/ligation-indepen...
Added: Sun Oct 25 2009, Hits: 2750, Reviews: 0 Write review Cached -
Ligase Independent Cloning (LIC) using pETM-13/LIC
(Dr. Arie Geerlof)
Creating a construct with a C-terminal His6-tag
http://www.embl-hamburg.de/~geerlof/protocols/pETM...
Added: Sun Oct 25 2009, Hits: 1438, Reviews: 0 Write review -
Ligase Independent Cloning (LIC) using pMyNT/LIC
(Dr. Arie Geerlof)
Creating a construct for the M. smegmatis expression system
http://www.embl-hamburg.de/~geerlof/protocols/pMyN...
Added: Sun Oct 25 2009, Hits: 1071, Reviews: 0 Write review -
Ligase independent cloning
(Dr. Arie Geerlof)
Ligase independent cloning (LIC) is a simple, fast and relatively cheap method to produce expression constructs. It makes use of the 3'--> 5'-activity of T4 DNA polymerase to create very specific 10-15 base single overhangs in the expression vector. PCR products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase as well. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very efficient because only the desired products can form.
http://www.embl-hamburg.de/~geerlof/protocols/LIC-...
Added: Sun Oct 25 2009, Hits: 2624, Reviews: 0 Write review