Propagating Gateway Vectors
Program in Systems Biology; Program in Molecular Medicine
Genetic Phenomena | Genetics and Genomics | Genetic Structures | Investigative Techniques | Laboratory and Basic Science Research | Molecular Biology | Systems Biology
Generating stocks of Entry and Destination vectors for use in the Gateway recombinatorial cloning system requires transforming them into Escherichia coli strain DB3.1, where they can replicate because this strain is immune to the effects of the ccdB gene carried in the Gateway cassette. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids will consequently allow growth of standard cloning strains of E. coli (e.g., DH5alpha). Therefore, after making new stocks of Gateway plasmids, their ability to grow in cloning strains of E. coli must be tested. This involves obtaining multiple stocks of vector, each arising from a single plasmid grown in a single DB3.1 bacterial colony, and transforming each stock into both DB3.1 and the preferred cloning strain of E. coli in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (i.e., no or few colonies are obtained after transformation) should be used in Gateway cloning reactions. The sequence can be performed in 3 d.
DOI of Published Version
Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094920. doi: 10.1101/pdb.prot094920. Link to article on publisher's site
Cold Spring Harbor protocols
Reece-Hoyes JS, Walhout AJ. (2018). Propagating Gateway Vectors. Program in Systems Biology Publications. https://doi.org/10.1101/pdb.prot094920. Retrieved from https://escholarship.umassmed.edu/sysbio_pubs/122