BIO230H1 Study Guide - Final Guide: Messenger Rna, Differential Centrifugation, Ribosomal Rna
42
BIO230H1 Full Course Notes
Verified Note
42 documents
Document Summary
In most cells rrna represents approximately 70-80%, trna 10-20% and mrna less than. In order to perform many fundamental molecular biology experiments, the first and often most crucial step is isolating high quality, intact rna. These experiments include northern hybridization, rt-pcr, nuclease protection assays, in vitro translation, cdna library construction, mircroarray analysis, molecular cloning, etc. Protocol: disrupt the cell wall and cell membrane enough to allow access to the rna (using lysozyme and sds), selectively precipitate the cellular constituents that you do not want (using. Sds and potassium acetate), leaving the rna in solution. Centrifuge to remove the precipitate: selectively precipitate the rna from the supernatant (using high salt and ethanol). Edta is a chelator, which has a very high affinity for mg2+. When divalent cations are all bound to the chelator, rnases requiring these ions become inactive. Sds (sodium dodecyl sulfate) is a strong ionic detergent that solubilized the plasma membrane.
