Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone
Program in Systems Biology; Program in Molecular Medicine
Genetic Phenomena | Genetics and Genomics | Genetic Structures | Investigative Techniques | Laboratory and Basic Science Research | Medical Sciences | Molecular Biology | Systems Biology
This protocol describes using the Gateway recombinatorial cloning system to create an Entry clone carrying an open reading frame (ORF) and then to transfer the ORF into a Destination vector. In this example, BP recombination is used to clone an ORF from a cDNA source into the Donor vector pDONR 221. The ORF from the resulting Entry clone is then transferred into the Destination vector pDEST-15; the product (the Destination clone) will express the ORF as an amino-terminal GST-fusion. The technique can be used as a guide for cloning any other DNA fragment of interest-a promoter sequence or 3' untranslated region (UTR), for example-with substitutions of different genetic material such as genomic DNA, att sites, and vectors as required. The series of constructions and transformations requires 9-15 d, not including time that may be required for sequence confirmation, if desired/necessary.
DOI of Published Version
Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094938. doi: 10.1101/pdb.prot094938. Link to article on publisher's site
Cold Spring Harbor protocols
Reece-Hoyes JS, Walhout AJ. (2018). Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone. Program in Systems Biology Publications. https://doi.org/10.1101/pdb.prot094938. Retrieved from https://escholarship.umassmed.edu/sysbio_pubs/121