Experiment 3 (exam).docx - Isolation Cloning Transformation...

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Isolation, Cloning & Transformation of Plasmid DNA (Experiment 3) Lab Manual Notes Antibiotic resistance - Ability of a microorganism to withstand the effects of antibiotic treatment - Bacteria can carry gene of resistance on their chromosome or an extrachromosomal piece of DNA such as a plasmid. Plasmids - Circular, double-stranded extrachromosomal DNA vehicles that can carry gene of resistance. - In this experiment: pUC18 & pKAN were used. o pUC18 : consists of pBR233-derived ampicillin resistance gene . o pKAN : contains kanamycin resistance gene . pUC18 - useful for cloning, sequencing and expressing foreign genes in E. coli. - It contains ampicillin resistance gene, origin of DNA replication (on portion of LacZ gene of E. coli). o If introduced into suitable E. coli host, strain carrying LacZ product will be able to complement portion on the plasmid thus cells will be able to make beta galactosidase (b gal). If b gal is produced, blue colonies will arise as it cleaves substrate X gal on plates. pKAN - If this plasmid is cloned into the multiple cloning site of pUC18, it will deactivate LacZ thus won’t be able to produce b-gal. o No b-gal = cannot breakdown X-gal = white colonies. In this experiment: - Isolate plasmids pUC18 & pKAN - Clone pKAN into MCS of pUC18 (deactivating LacZ gene) = recombinant plasmid . - Recombinant plasmid will be transformed into E. coli strain DH5a = multiresistant bacteria . o Appearance of white colonies on X-gal plates = indicate presence of kan gene inserted into pUC18 vector. Tutorial Notes Tutorial 4: Overall goal of this experiment is to transform a strain of DH5a E coli to be resistant to both ampicillin & kanamycin by creating recombinant pUC18 plasmid. pKAN plasmid - Carries the kanamycin resistance gene pUC18 plasmid - Carries the ampicillin resistance gene encoded as the beta-lactamase (bla) gene . - It also has origin of DNA (MCS) ligated to portion of LacZ gene.
Isolation, Cloning & Transformation of Plasmid DNA (Experiment 3) - If pUC18 is introduced to suitable E. coli host that also carries LacZ gene, it will be able to complement the portion of plasmid thus can make b-gal and produce blue colonies as it cleaves substrate X-gal. - X-gal o Chromogenic substrate. o Hydrolyzed by b-gal into colorless galactose + 4-chloro-3-bromoindigo, a blue precipitate. Experimental process : - Use agarose gel electrophoresis to confirm successful isolation of plasmids - Digest plasmids using restriction endonucleases o HindIII, BamHI - Ligate restriction fragment of pKAN into MCS of pUC18 in hopes to make a multiresistant recombinant plasmid.

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