Topic 6_Isolating_and_Separating_DNA Dr. Updike

Topic 6_Isolating_and_Separating_DNA Dr. Updike - Isolating...

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Isolating and Separating DNA Purified, precipitated DNA Cells containing DNA
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clinical diagnostics genetic mutations, viral infections recombinant DNA technology forensic science Why do we need to isolate DNA?
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DNA Isolation Break open the cell – capsule, cell wall, plasma membrane, nuclear envelope Separate DNA of interest from other cell components Protect DNA integrity without breaking (shearing) DNA and protecting from DNase
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Breaking open the cell Boil, freeze, or use mechanical disruption Alkaline lysis alkaline buffer consisting of a detergent (SDS) and a strong base. alkali conditions denature proteins & RNA (& large DNA, so not too long). This treatment is immediately followed by neutralizing with an acid. SDS (sodium dodecyl sulfate) disrupts the membrane bilayer and then coats broken cell walls, protein, & denatured components that can be precipitated from solution and away from your DNA of interest Enzymatic lysis lysozyme (bacterial cell walls) zymolyase (fungal cell walls) Proteinase K cleaves peptide bonds very resilient (works in wide pH ranges and temperatures) inactivates DNase & RNase
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Separating DNA of interest from other cellular components Properties of DNA we can use for its purification: large water soluble charged Separating DNA from RNA DNA is more unstable at low pH RNA is more unstable at high pH
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This document was uploaded on 11/04/2011 for the course MMBIO 240 at BYU.

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Topic 6_Isolating_and_Separating_DNA Dr. Updike - Isolating...

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