Ligation-independent cloning
Ligation-independent cloning (LIC) is a form of molecular cloning that can be performed without the use of restriction endonucleases or DNA ligase. The technique was developed in the early 1990s as an alternative to restriction enzyme/ligase cloning.[1] This allows genes to be cloned without the requirement of a restriction site for cloning that is absent from the gene insert.[2][3][4][clarification needed] LIC uses long complementary overhangs on the vector and the DNA insert to create a stable association between them. [5]
Steps in Procedure
[edit]- Design PCR Primers with LIC extension
- Perform PCR to amplify gene
- Purify PCR product
- Create 5' overhangs
- Incubate vector and PCR product to anneal
- Transform
References
[edit]- ^ "Seamless Cloning | NEB". www.neb.com. Retrieved 2020-01-23.
- ^ "Ligation Independent Cloning (LIC)". New England BioLabs (NEB). Retrieved 15 January 2016.
- ^ "Get Your Clone 90% Of The Time with Ligation Independent Cloning". Bitesize Bio. Retrieved 2012-05-09.
- ^ Haun, RS; Serventi, IM; Moss, J (1992). "Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors". BioTechniques. 13 (4): 515–8. PMID 1362067.
- ^ Snyder, Lori A. S. (2020). "Laboratory Techniques". Bacterial genetics and genomics. Boca Raton, FL: CRC Press. p. 301. ISBN 9781000039191.