Detecting, Quantifying & Resolving Nucleic Acids Flashcards
methods for DNA quantification
UV spectroscopy
Fluorometry
gel electrophoresis
Absorbance spectroscopy
goal is to measure the light of a specific wavelength absorbed by an analyte (chromophore)
Fluorescence spectroscopy
light emitted from an analyte (fluorophore) following the excitation by a light of a specific wavelength is measured
UV wavelengths
180-350 nm in length
used for nucleic acids
cannot use plastic cuvettes bc they will absorb this wavelength range
DNA maximal absorption
at 260 nm
dsDNA 1/extinction coefficient
50
RNA 1/extinction coefficient
40
ssDNA 1/extinction coefficient
33
DNA concentration calculation
absorbance @ 260 x 1/coefficient x dilution
most common DNA contaminant
proteins
maximal absorption of proteins
280 nm
260/280 ratio
> 1.6 to be able to use in downstream reactions
measure of protein contamination
Nanodrop
small UV spectrophotometer uses only 1 uL of DNA determines concentration & purity no dilutions does not tell about the quality of the DNA
Hoechst 333342/33258
fluorescent DNA binding dye
absorbance maximum 350 nm
emission maximum 461 nm (blue)
fluorescent RNA binding dye
PicoGreen or
RiboGreen
fluorometry pros & cons
pros: very quantitative, not influenced by contamination
cons: dyes are instable in light, says nothing about integrity of sample
Gel electrophoresis principle
negatively charged molecule will migrate towards the anode (+)
migration depends on size, charge & shape
gel electrophoresis can determine
presence of nucleic acid purity INTEGRITY quantify measure size/ length!
DNA charge
negatively charged due to the phosphodiester backbone
agarose gel
polysaccharide purified from seaweed
used as a molecular sieve
varying concentrations determine the resolution of the fragments of DNA
0.8% agar
500-3000 bp resolution
assessing genomic DNA integrity
large DNA fragments
2.0% agar
75-500 bp resolution
small PCR products
TAE
tris-acetate EDTA
cheap, easy to use
effective for running larger sized DNA fragments
TBE
tris-borate EDTA
2x buffering capacity of TAE
resolves small fragments better (
TAE & TBE functionality
both buffers bind divalent cations such that DNA has a uniform negative charge & runs strictly according to size
Ethidium Bromide
most commonly used agarose gel electrophoresis stain
added directly to the gel
sensitivity ~10ng of DNA
possible carcinogen
SYBR green or SYBR gold
gel electrophoresis stain cannot add directly to gel takes 30 min to stain sensitivity ~1ng DNA not toxic not used
PAGE
polyacrylamide gel electrophoresis
synthetic, consistent polymer
resolves 1 bp difference in a 1kb molecule
Capillary electrophoresis
separates solutes by charge/mass ratio
more rapid, automated than slab gels
RNA electrophoresis problem
RNA has a high degree of secondary structure & will not run in an electric field according to its length unless it is denatured
RNA electrophoresis
PAGE for sequencing/STR only (high resolution)
Automation
principles of electrophoresis & fluorescence
