DNA Analysis Flashcards
Preparation steps for DNA
Homogenisation/lysis
Purification
Precipitation/isolation
Homogenisation techniques depend on the organism, eukaryotes require breakdown of tissue.
Slurry containing nucleic acids and other macros, lot of lipids, require organic solvent to remove.
DNA Concentration by Absorbance
N base structure like benzene
So can absorb light due to delocalisation of electrons.
If proteins are not removed there will be absorbance at 280 as some amino acids have similar properties.
Polysaccharides can absorb light at 230.
Phenol absorbs at 230.
Beer law
A = 3/c/l
Absorbance
molar extinction coefficient
Concentration (units depend on 3)
Light path.
Standard coefficient for 1cm pathlength 50microg/ml
Electrophoresis
Movement of charged molecules in an electric field.
Phosphates are negatively charged, therefore DNA is also.
Preparation of electrophoresis gel
0.5-2% dissolved in buffer.
Add ethidium bromide to a conc of 0.5microg/ml.
Visible under UV
Electrophoresis, Cast gel
Heat the mix to dissolve the agarose, cool and pour into casting tray containing a comb to form the loading wells.
Running gel electrophoresis
Add loading dye to DNA samples.
Insert gel tray into apparatus and fill container with running buffer (TAE)
Load the DNA/dye samples into the wells.
Load a “marker”
Start the run (1-5V/cm)
Influences on DNA migration
Size of the DNA molecules
Agarose conc
DNA conformation
Voltage applied
Presence of ethidium bromide/type of agarose/electrophoresis buffer.
State of a plasmid (restricted vs unrestricted)
Calibration
need to calibrate to see how large DNA is, calibration depends on experiment conditions for every gel.
Run a ladder, shows the DNA presence in 1 line.
Fragment size (bp) against migration distance (MM) > calibration curve.
DNA quality
Good:
clear markers, one bright band.
Bad:
DNA degradation, shadow that spans.
RNA contamination in bottom.
Impurities at top.
