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IUB BIOL-L 211 - Molecular Biology Techniques II

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BIOL-L211 1st Edition Lecture 37 Outline of Last Lecture I. Techniques to examine DNA-protein interactionsII. EMSAIII. DNase footprinting assayIV. Chain-terminating method Outline of Current Lecture I. Molecular CloningCurrent LectureMolecular Biology Techniques III. Molecular CloningA. Molecular cloning: the ability to create new molecules of DNA that do not occur naturally and maintain (reproduce) them in cellsB. Recombinant DNA: molecules of DNA synthesized in a lab (that do not occur naturally)C. It is important to understand PCR (polymerase chain reaction) to understand molecular cloningD. Recall agarose gel electrophoresisE. Restriction endonucleases are EXTREMELY important for this technique1. Restriction endonucleases cut DNA into smaller pieces at specific sequences2. Restriction sites: sites 4-12 nucleotides long that are recognized by restriction enzymes (=restriction endonucleases)3. Used in labs to cut DNA into smaller pieces that can be analyzed by size by separation with agarose gel electrophoresis4. Restriction endonucleases produce a staggered cut, yielding a "sticky end" overhangF. Plasmid Vectors1. Required for cloningThese 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.2. Has an origin of replication (ori) (recall the number of oris depends on if a cell is eukaryotic or prokaryotic) that allows the plasmid to replicate independently of the cell's chromosome3. Has at least one gene coding resistance to specific antibiotics, which allows for the selection of the desired cells after replication4. Has one or more restriction sitesa. Multiple Cloning Site (MCS): cluster of restriction sites grouped together5. Serves as the backboneG. Insert1. One or more insert(s) are required for cloning2. Typically gene(s) of interest or a promoterH. Method:1. Use PCR to amplify the insert2. Apply the same restriction endonucleases to the plasmid vector and the insert3. Compatible sticky ends will form4. Combine the digested vector and insert: the pieces will connect like a puzzle5. DNA ligase joins the nicksI. Propagation (you want to reproduce more copies of this recombinant DNA)1. Introduce recombinant plasmid to E. coli by transformationa. Recall that transformation is the process by which some prokaryotes can take up genetic material from their environment in a state of "competence"2. Some (NOT ALL) of the bacteria will take up the plasmid3. The plasmid will replicate independently of the bacterial chromosome4. For transformation, bacteria are grown in a liquid culture5. After transformation, plate the bacteria on growth media with specific antibioticsa. The antibiotics will eliminate all bacteria that haven't taken up the plasmidb. All remaining cells have the desired


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