Isolation of biological macromoleculeTechnology 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

Example: Plasmid prepNeed to eliminate membranes, proteins, chromosomal DNA, and RNA

Lyse cells (rupture membranes) typically with alkaline lysisHigh pH and detergents will lyse cellsThen 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 complexAlkaline lysis solutions also contain Rnase which degrade RNA rapidly

Left with proteins and plasmid DNA in solutionLoad on spin column with nylon membrane

DNA will bind to nylon; presence of alcohol strengthens thisproteins will spin through membrane in the presence of alcohol

After this step, only plasmid DNA should be left on membraneadd water or buffer, let plasmid DNA leave membrane and spin through into

fresh tube

Variation on conceptual themeSame idea could play out with different steps

Boiling miniprep of plasmid DNACells are lysed by putting cells with lysozyme, then putting in boiling water

remove membranes and chromosomes by centrifugationRNA is degraded by Rnase

Proteins are removed by phenol extractionphenol is non-polar so proteins will tend to collect at polar/nonpolar interface of extraction

Collect aqueous phase which should have plasmid DNAPrecipitate plasmid DNA

DNA will precipitate in cold ethanol with NaCl presentPellet precipitated plasmid DNA by centrifugationDry and resuspend pellet in water or buffer

Same general concept of sequential exclusion, but specific mechanisms are different

Isolating chromosomal DNALyse 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 isopropanolplasmids will not make strands

Hook stranded DNA form precipitation and resuspend

Isolating RNALyse 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

Isolating ProteinsLyse 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