Purification of RNA and DNA Introducing DNA into cells May 2

Purification of DNA Plasmid DNA (1-6,9,15) –alkaline lysis –commercial kits DNA from mammalian cells (1-18) –cultured cells, tissues, blood DNA from yeast cells (12-8)

Concentrating DNA Ethanol precipitation SaltStock solutionFinal conc. ammonium acetate 10M M lithium chloride8M0.8M sodium chloride5M0.2M sodium acetate3M (pH 5.2)0.3M Add 2X volume of % ethanol

Purification of RNA RNA composition of a cell Ribonucleases (4-1)

Introduction of DNA into cells bacterial cells –competent cells (E.coli) (1-1) –electroporation (1-4) cultured mammalian cells –calcium phosphate (9-5) –electroporation –commercial lipid reagents –viral vectors

Today immunoblot of transferred proteins transfection of vero cells (to prepare positive and negative controls for quantitative real-time PCR)