杨荣武分子生物学课件Week2modification.pptVIP

DNA and RNA isolation, purification, visualization and quantitation Genomic DNA preparation overview Plasmid DNA preparation DNA purification Phenol extraction Ethanol precipitation RNA work Bacterial genomic DNA prep: cell extract Genomic DNA prep: removing proteins and RNA 2 ways to concentrate the genomic DNA Genomic DNA prep in plants -- how get rid of carbohydrates? Plasmid purification: alkaline lysis DNA purification: silica binding Visualizing DNA and RNA: non-specific methods Quantitation of DNA Electrophoresis Visualizing DNA in gels Visualizing DNA: Electrophoresis Allows separation of biomolecules (DNA, RNA, protein) on basis of size A separation matrix, or gel (agarose or polyacrylamide), is saturated with an electrically conductive buffer Samples are loaded, an electric field is applied, and negatively charged biomolecules in the sample travel toward the cathode The larger the molecule, the slower the travel through the gel matrix Dyes allow a visual estimate of the rate of travel through the gel The choice of matrix depends mainly on the size of DNA being analyzed Agarose gels Agarose: a polysaccharide polymer of alternating D- and L-galactose monomers, isolated from seaweed Pore size is defined by the agarose concentration (higher concentration, slower DNA migration overall) The conformation of the DNA (supercoiled, nicked circles, linear) affects the mobility of the DNA in gels Rate of DNA migration is affected by voltage (5 to 8 Volts/cm is close to optimal) Agarose comes in a myriad of types (variable melting temperatures, generated by differential hydroxyethylation of the agarose) Agarose gels Standard gels can separate DNA fragments from 100 bp to about 20,000 bp Pulsed-field gels separate very large DNA fragments (up to 10,000,000 bp, or 10 Mb) Polyacrylamide gels Acrylamide monomers (toxic!) polymerized to form gel matrix The gel structure is held together by the cross-linker-- usually N, N-methylenebisacrylamide (bis for short