measurements of the concentration of nucleic acids Flashcards
measurements of the concentration of nucleic acids (DNA and RNA)
measuring UV light absorbance Using
Spectrophotometer
fluorescence detection after use
fluorescence dye
absorption and consentration have a ….. relation
derictly
Solutions of nucleic acids strongly absorb ultraviolet light with
lmax of 260 nm
The intense absorption is due primarily to the presence of
aromatic rings in the purine and pyrimidine bases
On the other hand, the bases in single-stranded DNA or RNA are
More exposed and have more space between them
This increases their ability to absorb light
A solution of double stranded DNA at a concentration of
50 mg/ml in a 1-cm quartz cuvette will give an A260 reading of about 1.0
A solution of single-stranded DNA or RNA at a concentration of
40 mg/ml in a 1-cm quartz cuvette will give an A260 reading of about 1.0
UV light absorbance
The spectrophotometric assay for nucleic acids is
simple, rapid, nondestructive, and very sensitive.
UV light absorbance
It provides an indication of
structural changes
purity
The ratio of ……. is often used as a relative measure of the purity of nucleic acid solutions.
A260/A280
Pure DNA
has an absorbance ratio of about 1.8
Pure RNA
has an absorbance ratio of about 2
Fluorescence
is the phenomenon of the emission of radiation when the molecules are excited by radiation
fluorescence dyes are used in applications such as
ELISA assays, PCR, and DNA sequencing
