BIOC 3400 Quiz: Lab Quiz Summary
8 Mar 2017
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Purpose: to isolate and purify high molecular weight dna, high mw rna (26s+5. 8s rrna and 18s rrna) and low mw rna (trna and 5s rrna) from a eukaryote, what. Isolating pure nucleic acids is difficult because they are founf complexed with proteins: high mw as chromatin, rna in ribosomes. 3 methods to deproteinize cellular extracts: (1) with a highly purified protease solution degrades protein (2) strong salts to dissolve nucleic acid and precipitate denatured protein (3) us phenol as a protein denaturant. Must avoid nuclease degradation of dna and rna during extraction, purification and storage. Rna extraction often uses detergents (sds or sarkosyl) that can denartue ribonucleases. Chelating agents (edta) are often used to reduce nuclease activity. Phenol extraction yields 3 phases: (1) upper aqueous phase nucleic acids (2) lower phenol phase hydrophobic cell components (3) middle layer denatured protein. Precipitate the nucleic acid from the aqueous phase with absolute ethanol, the collect by centrifugation and rinsed with 80% ethanol.
