Marie Černá, Markéta Čimburová, Marianna Romžová

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Presentation transcript:

Marie Černá, Markéta Čimburová, Marianna Romžová 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 Phenol-chloroform extraction (different solubility conditions in solvents) Salting out method (protein precipitation by NaCl) Adsorption method (silica-gel membrane)

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

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

Gel Electrophoresis Separation method 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

Gel Electrophoresis Separation 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