Ligation-independent cloning (LIC) was developed for directional cloning of PCR products without restriction enzyme digestion or ligation reactions (Aslanidis 1990, Haun 1992). The LIC method takes advantage of the 3’ →5’ exonuclease activity of T4 DNA polymerase to create very specific 12 to 15 nucleotide single-stranded overhangs in the vector and the insert, so that the vast majority of annealed products consist of the desired molecules. The annealed LIC vector and insert are transformed into competent E. coli cells, and covalent bonds are formed at the vector-insert junctions within the cell to yield circular plasmid. Directional cloning of the insert is achieved with minimal non-recombinant background, and cloning is so efficient that virtually all of the resulting colonies contain the desired recombinant.

PCR products with complimentary overhangs are created by building appropriate 5’ extensions into the primers. The purified PCR products are treated with LIC-qualified T4 DNA polymerase in the presence of the appropriate dNTP to generate the specific vector-compatible overhangs. Please refer to the User Protocols for detailed primer design information.

Three Different Protease Cleavage Sites

Three families of multi-purpose LIC vectors are available: enterokinase (Ek/LIC), Factor Xa (Xa/LIC) and HRV 3C (3C/LIC). In each of these vectors, the LIC site enables the removal of all vector-encoded sequences from the target protein by digestion with either enterokinase, Factor Xa, or HRV 3C.

The Ek/LIC, Xa/LIC, and 3C/LIC vector kits provide the necessary reagents for creating single-stranded overhangs, annealing with the vector, and transforming competent E. coli cells. Each kit provides enough reagents for 20 annealings and transformations. A control insert is included to verify performance.  

Directional PCR cloning into the most powerful E. coli expression vectors

The pET-43.1 series of vectors are designed for cloning and high-level expression of peptide sequences fused with the 491 aa Nus•Tag™ protein. Vector encoded sequence can be completely removed by cleaving the Nus•Tag fusion protein with enterokinase. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the vector map (TB318). The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand.

Components:

•	1 µg	Ek/LIC Vector
•	10 µl	Ek/LIC Control Insert
•	25 U	T4 DNA Polymerase, LIC-qualified
•	50 µl	10X T4 DNA Polymerase Buffer
•	100 µl	100 mM DTT
•	50 µl	25 mM EDTA
•	40 µl	25 mM dATP
•	1.5 ml	Nuclease-free Water
•	22 × 50 µl	NovaBlue GigaSingles™ Competent Cells
•	0.2 ml	BL21(DE3) Competent Cells
•	0.2 ml	BL21(DE3)pLysS Competent Cells
•	5 × 2 ml	SOC Medium
•	10 µl	Test Plasmid