MBIO 3410 lecture Oct 9 2007

MBIO 3410 lecture Oct 9 2007 - Last Lecture - overview DNA...

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1 Last Lecture - overview DNA isolation Genomic DNA Purification Plasmid miniprep Alkaline lysis Plasmid purification Matrix plasmid purification Caesium chloride gradient Flow chart Ethanol extraction DNA constructions Recombinant construct Generating compatible sites Clone into a blunt-ended restriction site Using oligonucleotide linkers Directionality of recombination Transformations Chemical transformation Transformation by electroporation Transformation by gene gun Low transformation efficiency Increased transformation efficiency
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2 Clarification Supercoiled DNA binds less EtBr (topological constraints), due to it’s compact nature it is more dense and will separate lower on a CsCl gradient (similar to it’s separation in an agarose gel) Nicked and linear DNA can intercalate more EtBr, leading to extensive unwinding This causes the DNA to separate higher up in the CsCl gradient
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3 Selection of recombinants If all of the competent cells from the transformation were allowed to grow on an agar plate then millions of cells would result Because transformation is a relatively inefficient process most of the millions of bacteria that grow will not contain a plasmid molecule On a non-selective agar plate it is not possible to determine which (if any) of the bacteria contain a successfully transformed plasmid molecule
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4 A method for selection identifies clones containing a successfully transformed plasmid molecule This is almost always accomplished by the presence of an antibiotic resistance gene on the plasmid vector ex . β-lactamase ( bla ) gene conferring resistance to ampicillin if the transformed cells are grown on a plate containing ampicillin, only the cells that express β-lactamase due to the presence of transformed plasmid will survive Colonies that are formed on the surface of the agar have arisen from a single cell which contained the plasmid with an intact β-lactamase gene Non-selective plate Selective plate amp
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5 Growth of transformant for analysis Single colonies from the transformation plate are transferred to culture broth and grown to stationary phase Broth contains the antibiotic used to select the plasmid Plasmids can be lost from their host during prolonged periods without selection The plasmids can then prepared from the cultures using miniprep plasmid prep protocol www.rlc.dcccd.edu/. ../lab_manual/inoculation.jpg
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6 Gel analysis Recombinant plasmids can usually be distinguished from re- circulated vectors by the relative sizes of the plasmids, and further by the pattern of restriction digests non-recombination products E = Eco R1 U= uncut H= Hind III S= Sal I
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7 Analysis of a clone Once the clone has been identified as containing the gene of interest: Further investigation of the fragment by restriction mapping = the analysis of the fragmentation of DNA with restriction enzymes (old school) Sequencing the entire fragment to compare it to other
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This note was uploaded on 07/05/2008 for the course MBIO 3410 taught by Professor Richardson during the Fall '07 term at Manitoba.

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MBIO 3410 lecture Oct 9 2007 - Last Lecture - overview DNA...

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