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

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

3. 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)

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

7. 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

8. 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