MCDB 2150 1st Edition Lecture 20 Outline of Last Lecture - Gene expression- Chromatin structure- Post-transcriptional control- Post-translational modificationOutline of Current Lecture - Recombinant DNA technologyo How to clone a geneThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Molecular Analysis & BiotechnologyRecombinant DNA technology = isolating & manipulating DNA using two sourcesHow to clone a gene:1.) Isolate gene of interest (your fav gene = YFG)a. PCR2.) Digest YFG & cloning vector (plasmid) with restriction enzymesa. These plasmids have unique restriction sites = multiple cloning sites (MCS site)b. ORI site = origin of replicationc. An antibiotic resistance gene (like ampR) – allows for selection of transformed cellsd. Restriction enzymes cut palindromes and create sticky ends3.) Mix and bind together foreign & plasmid DNAa. Cut with same restriction enzymes (sticky ends!!!)b. DNA ligase seals the nick in the backbone4.) Introduce plasmid into bacterial cellsa. Competent bacteria (chemically treated to uptake plasmid)i. Transformation is still rare sooo…5.) Select for transformed cellsa. The plasmid contains an antibiotic resistance geneb. Grow the cells in the presence of this antibiotic and only the antibiotic resistant cells will survive6.) Produce recombinant protein –
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