MBB 222 Lecture Notes - Lecture 22: Pbr322, Multiple Cloning Site, Ampicillin
21 Mar 2016
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Dna cloning - separating a specific gene or dna segment from a larger chromosome, attaching it to carrier dna (a vector), and replicating it thousands or millions of times. Dna purification: lyse cells, centrifuge down cellular debris, remove protein (phenol extract, cscl gradients, concentrate dna by ethanol precipitation (dna is insoluble in ethanol, treat with ribonucleases to remove rna. Restriction endonucleases s are dna scissors that recognize and cleave double stranded dna at specific sequences: most restriction recognition sequences are 4-6 bp long (some are 8 bp) that are palindromic. Found in a wide range of bacterial species and its natural function is to cleave foreign dna (like an infecting phage): bacterial dna is methylated by a bacterial methylase to identify the dna as bacterial. **it is implicit that the 5" end is on the left in all such representations. Popular method involves carrying out single and double digests and comparing the types of products generated by agarose gel electrophoresis.
