AAT Bioquest

How many restriction enzymes are used to insert a specific DNA sequence into a vector in molecular cloning?

Posted July 22, 2020


Two restriction enzymes are needed. Both the DNA sequence to be inserted and the vector have to be treated with these two restriction enzymes. Each restriction enzyme generates a pair of compatible ends on the DNA and vector, respectively, which are then joined and covalently sealed by DNA ligase, thus inserting the DNA sequence of interest into the vector. To ensure the right orientation of insertion, at least one of the enzymes used should generate a sticky end.

Additional resources

Helixyte™ Green *10,000X Aqueous PCR Solution*

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Kessler, C., & Manta, V. (1990). Specificity of restriction endonucleases and DNA modification methyltransferases—a review (Edition 3). Gene, 92(1-2), 1-240.

Brownlee, C. (2005). Danna and Nathans: Restriction enzymes and the boon to modern molecular biology. Proceedings of the National Academy of Sciences, 102(17), 5909-5909.