MBIO 3410 lecture Oct 4 2007

MBIO 3410 lecture Oct 4 2007 - Last Lecture - overview...

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1 Last Lecture - overview Pulsed-field Gel Electrophoresis Field inversion gel electrophoresis (FIGE) Contour-clamped homogeneous electric field (CHEF) electrophoresis Genetic engineering DNA cloning - overview Hosts Cloning Vectors - overview Introduction to plasmid vectors DNA libraries Genomic libraries cDNA libraries Screening libraries Overview of subcloning DNA isolation Genomic DNA Purification
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2 DNA isolation
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3 Plasmid miniprep Vector plasmids are smaller than E. coli chromosomal DNA They can be separated from the chromosomal DNA by physio-chemical methods: Alkaline lysis takes advantage of the fact that at alkali pH (between 12.0 and 12.5) linear DNA, but not closed- circular DNA, will become denatured Most methods to purify plasmid DNA based on this observation require small cultures of bacterial cells (1–5 mL of bacterial culture) and can yield up to 10 μg of purified plasmid DNA in a typical mini-prep procedure
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4 Alkaline lysis Most common method for purification of plasmid DNA from chromosomal DNA A bacterial culture can be centrifuged to pellet the cells The pellet is resuspended in a buffer that contains lysozyme to digest the cell wall of the bacteria Cell lysis solution is added Sodium dodecyl sulfate (SDS) detergent SDS disrupts the cell membrane and denatures the proteins alkaline sodium hydroxide solution is added Denatures the DNA and begins hydrolysis of RNA The preparation is neutralized with a concentrated solution of potassium acetate at pH 5 Precipitates the denatured proteins and chromosomal DNA and most of the SDS (insoluble in water) The preparation is centrifuged again, the supernatant ( lysate ) contains plasmid DNA, small RNA and some protein
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5 Plasmid purification The plasmid can be purified by selective binding to a positively charged (anionic) ion exchange matrix or membrane which allows the RNA and protein to be washed away or The classic method extracting the lysate with phenol or phenol-chloroform mixture (see ‘Genomic DNA Purification’)
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6 Fig 2.25 Matrix plasmid purification Plasmid DNA is bound to a silica matrix in buffer containing high levels of salt Under these conditions, other contaminants will not bind to the matrix and can be washed away Subsequently, the DNA can be eluted from the matrix using a low-salt buffer (or water) Larger-scale plasmid preparations procedures will often bind the plasmid DNA to a positively charged ion-exchange resin, such as DEAE (diethylaminoethanol), in buffer containing 1 M NaCl This level of salt allows the DNA to bind to the matrix, but not the contaminants After washing, the salt concentration is raised to 1.6 M to elute the DNA The DNA is precipitated with ethanol and resuspended in a low-salt buffer before it can be used in many molecular biology protocols
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7 Caesium chloride gradient Caesium chloride (CsCl) forms a uniform density gradient
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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 4 2007 - Last Lecture - overview...

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