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BIOL 239
Christine Dupont

Set 153 Type II REN restriction endonucleasetype II RENs recognize inverted palindromic sequences12 REN digestion of plasmid DNALane 1 is a size marker The rest are cut with the same or different enzymesWhat were staining with tends to stain double stranded things like DNA but not always DNA Not a discrete size theyre quite smallThe glowing bobs are form hydrogen bonds like DNA tRNA molecules linked to proteins and forms secondary structurePeople use RNAse which degrades RNA so you dont see it It doesnt make a difference14 Restriction MapsShow the order of different size fragments within a clone Consist of linear or circular diagrams of the sites along a DNA segment at which one or another restriction enzyme cleaves the molecule16 Molecular Cloning DNA fragments DNA cloningThe process of using living cells to make many exact replicas of a fragments of foreign DNA17 Molecular Cloning1 take a DNA fragment and insert the fragment into a specialized chromosomelike carrier termed a vector2 transfer the vector with its insert into cells host like Ecoli Copies will be made through replication Ecoli treats it like it is its own These are therefore known as DNA clones18 types of vectorsPlasmidsusually circular doublestranded DNA Each has an origin of replication several unique sites for restriction enzymes and a gene referred to as a selectable marker engineered to have these elements added by humans20Plasmid bluescript II aka pBluescript II21 transform the ligation mix into EcoliProcess of taking up a plasmid is transformationWeve cut the vector isolated fragment of interest and added ligase Ideally we want the construct shown in the last slide but in reality the fragment of interest can ligate with itself Why would it just go into the vector You have to select for the target construct
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