MCB 2050 Study Guide - Midterm Guide: Multiple Cloning Site, Reverse Transcriptase, Lac Operon
19 Oct 2014
School
Department
Course
Professor
Document Summary
Topic 1, lectures 1 3: techniques of molecular genetics: basic techniques used to clone genes. Cloning steps: digest vector and dna of interest, anneal/ligate, transform e. coli, selection) Restriction enzymes: function in bacteria degrade invading dna, bacteria must methylate their own dna to prevent digestion by their own restriction enzymes (re), blunt vs staggered cut\ Cloning vectors (plasmids): 3 essential components: origin of replication, dominant selectable marker (antibiotic resistance gene), unique re sites for gene insertion. Screening for transformants: bluescript blue/white screening, mcs in lacz coding region. Spread transformants on agar with x-gal: blue colonies (lacz intact no insert), white colonies (lacz gene disrupted insert present) Mammalian expression vectors: bacterial ori and selection, eukaryotic promoter, mcs (multiple cloning site), polyadenylation signal, eukaryotic selection. Construction and screening of dna and cdna libraries: complementation, hybridization. Principle: forward and reverse primer, one to amplify each strand, products doubled with each reaction.
