BIOL 200 Lecture Notes - Lecture 22: Molecular Cloning, Dna Ligase, Recombinant Dna
Document Summary
To prepare large quantities of identical dna: vector + dna fragment = insert, recombinant dna (any dna molecule derived from different sources, replication within host cells, isolation, sequencing, manipulation of purified dna fragment. Hundreds of restriction enzymes each recognizing a specific target sequence and making a specific cut. Dna cloning: joining of two molecules having compatible termini by dna ligase. Plasmid vector + dna fragment to be cloned recombinant plasmid: transformation and selection. Recombinant plasmid + e. coli plasmids in presence of cacl2 (heat pulse) culture on nutrient agar plates containing ampicillin. Cells that do not take up plasmid die on ampicillin plates. Dna libraries cdna = complementary dna ie. complementary to an mrna sequence: initially single stranded but easily converted to double-stranded. Genomic dna libraries: mixture of chromosomal dna fragments clone in vector bacteriophages/cosmids (propagated by infecting bacteria or in bacs. Need a promoter to drive transcription of the inserted protein-coding dna.