杨荣武分子生物学课件Week1.pptVIP

Molecular Biology Techniques Instructed by Professor Robert Young My grading policy Two pop quizzes (10%) One presentation (30%) Final exam (40%) 2006: 53 years of DNA structure Structure of DNA DNA is easy to work with… Readily isolated--plasmid isolation, PCR Stable--not chemically reactive like RNA (even archaeologically stable!) Easy to propagate and move from cell to cell Easy to make specific constructs Easy to make specific mutations Very easy to sequence (record-keeping) Predictable behavior Sequence lends itself to analysis--genome projects Visualizing DNA (and RNA, protein): non-specific detection methods Quantitation of DNA Electrophoresis Visualizing DNA ( protein) in gels Quantitation of DNA by UV absorbance 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) Polyac