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DNA Isolation

DNA Isolation. Marie Černá, Markéta Čimburová, Marianna Romžová. DNA Isolation Basic Steps. Cell lysis Removal of proteins - protease - adsorption or extraction DNA precipitation by ethanol DNA dilution in water or buffer. DNA Isolation Three Basic Methods.

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DNA Isolation

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  1. DNA Isolation Marie Černá, Markéta Čimburová, Marianna Romžová

  2. DNA IsolationBasic Steps • Cell lysis • Removal of proteins - protease - adsorption or extraction • DNA precipitation by ethanol • DNA dilution in water or buffer

  3. DNA IsolationThree Basic Methods • Phenol-chloroform extraction (different solubility conditions in solvents) • Salting out method (protein precipitation by NaCl) • Adsorption method (silica-gel membrane)

  4. DNA Purity and Concentration Spectrophotometry Absorbance maximum for nucleic acids 260 nm for proteins 280 nm • Concentration of DNA – at 260 nm • Purity of DNA: ratio of 260/280 nm

  5. DNA Purity and Concentration Fluorescent dyes • DNA is stained by intercalating dyes in gel (ethidium bromide) • Gel is loading with DNA standard (its concentration is pre-evaluated) • Comparison of two light intensities – standard and our sample

  6. Gel ElectrophoresisSeparation method Gel – sieve structure of polymer molecules with pores Gel - agarose - polyacrylamid Ethidium bromide is an intercalating dye, binds to DNA It creates complex exciting photons after UV-exposure

  7. Gel ElectrophoresisSeparation method PRINCIPLE: • the movement of charged molecules in electric field • the movement direction from – to + (the nucleic acids consist of negatively charged phosphate groups) • DNA-rate in gel depends on DNA-fragment length in indirect proportion The length of unknown fragments is compared to the length of standard fragments

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