Isolation of biological macromolecule Technology to simply go into a mixture and grab a single type...
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Transcript of Isolation of biological macromolecule Technology to simply go into a mixture and grab a single type...
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