DNA isolation-英文.pptVIP

Isolation of Nucleic Acids Goals: removal of proteins DNA vs RNA isolation of a specific type of DNA (or RNA) Types of Methods: differential solubility ‘adsorption’ methods density gradient centrifugation Types of DNA: genomic (chromosomal) organellar (satellite) plasmid (extra-chromosomal) phage/viral (ds or ss) complementary (mRNA) General Features: denaturing cell lysis (SDS, alkali, boiling, chaotropic) ? enzyme treatments protease RNase (DNase-free) DNase (RNase-free) * Dr.Saba Abdi High MW Genomic DNA Isolation Typical Procedure Cell Lysis 0.5% SDS + proteinase K (55o several hours) Phenol Extraction gentle rocking several hours Ethanol Precipitation RNAse followed by proteinase K Repeat phenol extrac-tion and EtOH ppt Phenol Extraction mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/isoamyl alcohol extraction(s) ? aqueous phase (nucleic acids) ? phenol phase (proteins) * Dr.Saba Abdi High MW Genomic DNA Isolation Typical Procedure Cell Lysis 0.5% SDS + proteinase K (55o several hours) Phenol Extraction gentle rocking several hours Ethanol Precipitation RNAse followed by proteinase K Repeat Phenol Extrac-tion and EtOH ppt EtOH Precipitation 2-2.5 volumes EtOH, -20o high salt, pH 5-5.5 centrifuge or ‘spool’ out * Dr.Saba Abdi Isolation of RNA Special Considerations RNAse inhibitors! extraction in guanidine salts phenol extractions at pH 5-6 (pH 8 for DNA) treatment with RNase-free DNase selective precipitation of high MW forms (rRNA, mRNA) with LiCl oligo-dT column * Dr.Saba Abdi Plasmid Miniprep Protocol 1. Solubilize bacteria in alkali solution 2. Neutralize with Na-acetate 3. Centrifuge, discard pellet 4. Mix supernatant with resin + chaotropic agent 5. Wash resin 6. Elute DNA with low salt buffer Adsorption Methods nucleic acids selectively absorb to silica or resins in the presence of certain chaotropic agents or salts applications: plasmid preps fragments after electrophoresis PCR templates * Dr.Saba Abdi Densit