MIDTERM LEC: MEASUREMENT OF NUCLEIC ACID QUALITY & QUANTITY Flashcards
WHAT ARE THE 4 METHODS MEASUREMENT OF THE QUALITY & QUANTITY OF DNA & RNA???
ELECTROPHORESIS
SPECTROPHOTOMETRY
FLUOROMETRY
MICROFLUIDICS
Separation of
particles through a solution or
matrix under the force of an
electric current
ELECTROPHORESIS
Technique that uses light absorption to measure the concentration of an analyte in solution
SPECTROPHOTOMETRY
Measurement of emitted fluorescent light
FLUOROMETRY
Science & technology of systems that process or manipulate small
amounts of fluid, using channels measuring from tens to hundreds of micrometers
MICROFLUIDICS
▪ DNA & RNA can be analyzed for quality (detection & size analysis) by resolving an aliquot of the isolated sample on an agarose gel
▪ Uses an electric current to propel charged biomolecules through a porous gel matrix at a rate that is the function of the charge, size, & shape of the molecules
ELECTROPHORESIS
Fluorescent dyes used in ELECTROPHORESIS
ethidium bromide, SybrGreen I & II, silver stain
Appearance of DNA on agarose gels depends on the type of DNA isolated
ELECTROPHORESIS
Nucleic acids absorb light at 260 nm through the adenine residues
SPECTROPHOTOMETRY
Beer-Lambert Law:
A = ∈bc
A = ∈bc
❖ A = absorbance
❖ ∈ = molar absorptivity (L/mol-cm), 50 for dsDNA, 40 for RNA
❖ b = path length (cm)
❖ c = concentration (mg/L)
Absorbance is _____ proportional to the concentration of the nucleic acid in the sample
directly
molar absorptivity for DNA???
50 mg/L or ug/mL
molar absorptivity for RNA???
40 mg/L or ug/mL
Using absorptivity, as a conversion factor from optical density to concentration:
At 260 nm, 1 optical density unit (or absorbance unit) = 50 mg/L (or 50 μg/mL) of dsDNA & 40 μg/mL of RNA