Ligase Independent Cloning (LIC)

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.

  1. Preparation of vector DNA

The EMBL-made LIC vectors (see appendix for vectors maps) all contain the gene encoding for eGFP flanked by two BsaI sites (shown in red). These sites are used to linearize the vector, while at the same time removing the eGFP gene.

ATTTTCAGGGCGCCATGAGACCG..eGFP..GGTCTCACCGCGTCGGGTCACCAC

TAAAAGTCCCGCGGTACTCTGGC..eGFP..CCAGAGTGGCGCAGCCCAGTGGTG

 

BsaI

V

 

ATTTTCAGGGC………………………CCGCGTCGGGTCACCAC

TAAAAGTCCCGCGGT………………………CAGCCCAGTGGTG

Next the digested vector is treated with T4 DNA polymerase in the presence of dTTP. Because of the 3′–> 5′ activity of the polymerase the bases are removed from both 3′-ends until the first thymidine (T) residue is reached.

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