Biology Reference
In-Depth Information
both DNA and RNA if an RNase has not been used to eliminate the RNA, is
placed into a quartz cuvette that is then placed into the UV spectrophotometer.
UV light is passed through the sample at a specified path length, and the absor-
bance of the sample at specific wavelengths is measured (A
260
, or absorbance at
260 nm and A
280
) to measure contaminating protein in the sample. Based on the
readings, the concentration of the sample is determined and the A
260
/A
280
ratios
are calculated to indicate sample purity. The maximum absorbance of nucleic
acids occurs at 260 nm, and DNA should yield an absorbance ratio of
>
1.8, with a
maximum of 2.0. The A
260
/A
230
ratio is a key measure of purity. A
230
is the wave-
length that can measure contaminants such as chaotropic salts, which can inhibit
PCR, and the A
260
/A
230
ratio should be
>
1.4, on a scale of 2. UV spectrophotom-
etry is common and simple and can only detect concentrations of nucleic acids
of
≥
1.5
μ
g/ml. The presence of proteins, RNA, and chaotropic salts can result in
false estimates of DNA concentration, as can Tris buffer, EDTA, and GuSCN salts.
The presence of free nucleotides and changes in pH also affect quantitation of
DNA (
(Wilfinger et al. 1997
).
An improved version of UV spectrophotometry is available in the NanoDrop
series of equipment. It requires only 1
−
2
μ
l of sample, which is retained on an
optical fiber that assesses the UV absorbance of the sample between 220 and
750nm. Software provided with the machine allows analysis of small samples.
The concentration of the sample and its entire absorbance spectrum are pro-
vided in graphical form, thereby allowing contaminants to be detected and iden-
tified based on their absorbance wavelengths. The NanoDrop technology can
evaluate a wide range of sample concentrations (
Boesenberg-Smith et al. 2012
).
Another method for estimating nucleic acids quantity is fluorometric. Dyes,
which intercalate and bind nucleic acid grooves, bind nonspecifically or selec-
tively to nucleic material. Ethidium bromide is one fluorometric dye that binds
double-stranded DNA by intercalation, but it has a high level of natural fluores-
cence so it is limited in its sensitivity. Other fluorometric methods involve the use
of Hoeschst 33258 and PicoGreen dyes. These methods require specific instru-
ments and a variety of reagent kits.
5.4 Precipitating Nucleic Acids
During cloning, it is often necessary to concentrate DNA samples or change the
solvent in which the nucleic acid is dissolved. DNA isolated by phenol contains
trace amounts of phenol that could disrupt the activity of enzymes in subse-
quent manipulations if it were not purified further. Purification can be achieved
