基因,蛋白,免疫 课程1.ppt

* Extraction, Manipulation, Cloning and Analysis of Nucleic Acid Genomics 1 Extraction of Nucleic Acids Molecular Biologists, at different times, will need to prepare several different kinds of nucleic acid. These include: DNA Cellular DNA Recombinant DNA Plasmid DNA Bacteriophage DNA RNA Total RNA Messenger RNA Nucleic Acid Extraction DNA Gentle lysis in the presence of : EDTA ( to inhibit DNases). Ionic detergent (to denature and remove proteins bound to DNA). Proteases to digest cellular proteins to peptides. Treatment with DNase-free RNase to degrade RNA. Gentle extraction (avoiding shear) with organic solvent to remove degraded RNA and peptides. Dialysis, spooling or ethanol precipitation. Quantitation and quality control Quantitation from OD@ 260 mn (1 mg/ml has OD of 20). Quality control from: Spectrum (OD@260 nm should be 1.8 fold greater than at 280 nm). Agarose gel electrophoresis for size of fragments. Nucleic Acid Extraction RNA Particular problems associated with extraction of RNA RNA is susceptible to degradation by RNases RNases are ubiquitous and difficult to to inactivate. Work in “RNase-free” environment. Autoclaved solutions. Sterile tips. Gloves. Heat sterilised glassware All solutions made with diethyl- pyrocarbonatetreated water. Quantitation from OD@ 260 mn (1 mg/ml has OD of 24). Quality control from: Spectrum (OD@260 nm should be at least 2 fold greater than at 280 nm). Purification of messenger RNA The majority of RNA in a cell is ribosomal RNA and transfer RNA. Messenger RNA constitutes about 4% of total RNA Almost all methods rely on affinity chromatography which exploits the presence of a poly(A) tail at the 3’ end of mRNAs. Immobilised Oligo (dT) Extraction of plasmid DNA Requires purification of plasmid DNA (small) from bacterial chromosomal DNA (large) Treatment with alkali-denatures chromosomal DNA but not small supercoiled plasmid DNA. Precipitation in high salt-precipitates singl