Isolation of biological macromolecule

1. Isolation of biological macromolecule Technology to simply go into a mixture and grab a single type of molecule is not readily available Instead use procedures to eliminate or exclude other types of molecules leaving the desired one behind Achieved by taking advantages of differences between molecules

2. Example: Plasmid prep Need to eliminate membranes, proteins, chromosomal DNA, and RNA Lyse cells (rupture membranes) typically with alkaline lysis High pH and detergents will lyse cells Then neutralize pH which causes membranes to clump Chromosomal DNA stays attached to membranes unless it is sheared by rough pipetting or vortexing Centrifuge to pellet membrane/chromosome complex Alkaline lysis solutions also contain Rnase which degrade RNA rapidly Left with proteins and plasmid DNA in solution Load on spin column with nylon membrane DNA will bind to nylon; presence of alcohol strengthens this proteins will spin through membrane in the presence of alcohol After this step, only plasmid DNA should be left on membrane add water or buffer, let plasmid DNA leave membrane and spin through into fresh tube

3. Variation on conceptual theme Same idea could play out with different steps Boiling miniprep of plasmid DNA Cells are lysed by putting cells with lysozyme, then putting in boiling water remove membranes and chromosomes by centrifugation RNA is degraded by Rnase Proteins are removed by phenol extraction phenol is non-polar so proteins will tend to collect at polar/nonpolar interface of extraction Collect aqueous phase which should have plasmid DNA Precipitate plasmid DNA DNA will precipitate in cold ethanol with NaCl present Pellet precipitated plasmid DNA by centrifugation Dry and resuspend pellet in water or buffer Same general concept of sequential exclusion, but specific mechanisms are different

4. Isolating chromosomal DNA Lyse cells with detergent at neutral pH and chromosome will not adhere to membranes Degrade RNA with Rnase Remove proteins with proteinase and/or by phenol extraction Chromosomal DNA will make long strands when precipitated in isopropanol plasmids will not make strands Hook stranded DNA form precipitation and resuspend

5. Isolating RNA Lyse cells and remove membranes by centrifugation Degrade DNA with Dnase enzyme Remove proteins by protease and/or phenol extraction Precipitate RNA in very cold ethanol precipitation or on column

6. Isolating Proteins Lyse cells and remove membranes by centrifugation Degrade RNA with Rnase Degrade DNA with Dnase Precipitate protein with ammonium sulfate Suspend and dialyze to remove excess salt