From Bacteria
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Bacteria are grown and harvested Cells broken open to release contents Cell extract treated to remove all components except DNA DNA solution is concentrated
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Bacteria are grown and harvested Cells broken open to release contents Cell extract treated to remove all components except DNA DNA solution is concentrated
Defined Medium
Components (quantities) are known Inorganic elements, vitamins, etc. added Example: M9
Exact components are not known Contains tryptone and yeast extract, which are mixtures of unknown chemicals Example: LB
Harvesting Cells
Mechanical force breaks cell membrane/wall Mortar and pestle; sonication Not usually used for DNA prep
Attack cell wall and cell membrane Cell wall = lysozyme, EDTA or both Cell membrane = detergent (SDS)
Lysozyme digests polysaccharides structural components of cell wall EDTA (ethylenediamine tetraacetate) binds magnesium ions essential for preserving structure of cell and inhibits enzymes that could degrade DNA
Purification of DNA
In addition to DNA, a large amount of proteins and RNA remain. These must be removed to avoid interference with further analysis
Organic Extraction
Add phenol or phenol/chloroform Proteins are precipitated; form white layer at interface between organic and aqueous layers. Aqueous solution removed
Organic Extraction
Organic Extraction
What if there are excessive proteins?
Pronase or proteinase K
Organic Extraction
Some mRNA removed with phenol treatment. Remaining RNA can be digested with ribonuclease (if necessary)
Concentration of DNA
If a small amount of DNA is targeted, the resulting solution will be dilute. One method is precipitation by ethanol. Precipitate centrifuged; supernatant removed.
Concentration of DNA
Silica Extraction
Silica added directly; or sample passed through silica column. DNA removed by adding water
Lysozyme has no effect Cetyltrimethylammonium (CTAB) added; binds nucleic acid and precipitates Centrifuged; supernatant removed
Centrifuge
Ultracentrifuge
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Bacteria are grown and harvested Cells broken open to release contents Cell extract treated to remove all components except plasmid DNA DNA solution is concentrated
Lysozyme and EDTA treatment in presence of sucrose prevents immediate burst. Sphaeroplasts formed; cell can be lysed with addition of non-ionic detergent.
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Narrow pH range where supercoiled DNA is denatured and regular DNA is not. Clear lysate is usually used. Base added; regular DNA is denatured Acid added; regular DNA tangled mass Tangled mass pelleted
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Supercoiled DNA also separated from regular DNA using ethidium bromide coupled with a caesium chloride (CsCl) density gradient.
Ethidium bromide will bind normal DNA, but only a limited amount of supercoiled DNA. This binding causes a shift in band of normal DNA.
Bacteriophage DNA
Phage DNA can be outside the cell; do not have to start
Must ensure phage is in lytic phase cI gene keeps phage in lysogenic phase cIts (temperature sensitive) mutants will enter lytic phase at 42 C
Phages must infect bacteria at just the right time in the growth cycle.
Links
http://learn.genetics.utah.edu/content/labs/extractio
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