The traditional methods of inserting genes into vectors are based on DNA cleavage by restriction endonuclease and then ligation by DNA ligase. However, this approach to DNA engineering is time-consuming and relatively inefficient. Ligation Independent Cloning (LIC) technique is developed as an alternative to restriction endonuclease/ligase cloning. LIC provides a simple and cost-effective tool for producing many constructs of a single target or multiple targets in parallel without the need to select specific restriction enzymes for each gene.
T4 DNA polymerase is a template-dependent DNA polymerase that has 3’-exodeoxyribonuclease activity, but lacks 5’ to 3’ exodeoxyribonuclease activity. LIC uses the 3´-exodeoxyribonuclease activity of T4 DNA polymerase to assemble DNA molecules by non-covalent complementation of single-stranded DNA of the insert and vector. Briefly, LIC relies on about 10- to 15-nucleotide complementary 3′ overhangs at the ends of a PCR-amplified DNA fragment and a linearized vector to make a stable hybridization product that can be readily used to transform host organisms without ligation. LIC based on T4 DNA polymerase cloning is strictly sequence-dependent as it requires the presence or absence of specific nucleotides at certain locations in the overlapping region.
pColiExpress™ LIC Cloning & Expression Kits are a highly efficient, versatile and fast system of DNA cloning vectors for protein expression in E. coli. All family systems are based in directional LIC technology, a rapid procedure that provides high-cloning efficiency and the fast production of a large quantity of any desired proteins.
|Product Name||pColiExpress™ I||pColiExpress™ II||pColiExpress™ III||pColiExpress™ IV|
|HRV protease cut site||√|
|Low copy number (ori pBR322)||√||√||√||√|
|BL21 (DE3) pLys||√||√||√||√|
|pColiExpress™ I Cloning & Expression Kit||
|pColiExpress™ II Cloning & Expression Kit|
|pColiExpress™ III Cloning & Expression Kit|
|pColiExpress™ IV Cloning & Expression Kit|
|BL21 (DE3) Competent Cells||Protein Expression using the T7 Expression System.|