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DNA Quantification

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31 views

DNA Quantification

Uploaded by

Kanishk Agrawal
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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NUCLEIC ACID QUANTIFICATION USING UV ABSORPTION SPECTROSCOPY

Aim: Quantification of DNA using UV Absorption Spectroscopy

Theory:

The traditional method for determining the amount of DNA in solution is by measuring
absorbance at 260 nm. The UV absorption of DNA and RNA (with a maximum at 260 nm) is
due to the purine and pyrimidine bases of the nucleotide components of the DNA.

When light passes through a sample, the intensity of the light emerging from the sample is
decreased. The transmittance T is defined as

T = I/Io, (1)
Where, I = Intensity of emerging beam
Io = Intensity of incident beam

To study the quantitative absorption of radiation by samples, it is useful to define another


quantity, the absorbance A

A = log ( Io/ I) = log (1/T) = − log T (2)

A linear relationship exists between the Absorbance A and the concentration c of the
absorbing species in the sample. Absorbance is proportional to path length l and
concentration c.

Thus, A = εcl, (3)

where the proportionality constant ε is called the molar absorptivity which is a measure of
the ability of the absorbing species in the sample to absorb light at the particular wavelength
used.

Equation (3) is known as the Beer-Lambert’s Law. Measuring the absorbance of a sample
can thus allow us to find out its concentration.

Some of the drawbacks are that relatively large amounts of DNA are required to get accurate
readings. Furthermore, the method cannot discriminate between RNA and DNA, and UV-
absorbing contaminants such as protein will cause discrepancies. However, because many
potential contaminants of DNA and RNA preparations absorb in the UV range at different
peak values, absorption spectroscopy is a reliable method for assessing both the purity of a
preparation and the quantity of DNA or RNA present using a ratio of OD260/OD280.
PROTOCOL: NUCLEIC ACID QUANTIFICATION
Absorbance measurements (A260) are straightforward as long as any contribution from
contaminants and the buffer components are taken into account. A reading from a reagent
blank (containing no nucleic acids) is taken prior to adding the nucleic acid. In instruments
where the readout can be set to indicate concentration, a known concentration is used for
calibration and subsequent readings are expressed as μg/ml, ng/ml, or pg/ml of nucleic acids.
Absorption of the sample is measured at several different wavelengths to assess purity and
concentration of nucleic acids. While absorbance readings cannot discriminate between DNA
and RNA, A260 measurements are quantitative for relatively pure nucleic acid preparations in
microgram quantities.

Materials
• 1× TE buffer or MQ water
• DNA sample to be quantitated in 1× TE buffer or MQ water (see recipe)
• Matched quartz semi-micro spectrophotometer cuvettes (1-cm path length)
• Single- or dual-beam spectrophotometer with ultraviolet to visible light source

NOTE: Sample cuvettes should be rinsed and drained of any liquid to eliminate carryover
prior to adding the subsequent sample.

For single-beam spectrophotometers


1. Pipette 1.0 ml of 1× TE buffer/MQ water into a quartz cuvette.
2. Place the cuvette in a single-beam spectrophotometer, take a reading at 325 nm and
zero the instrument.
Always hold the cuvettes by the corners only and wipe clean with optical tissue between
readings, if necessary
3. Remove blank cuvette and insert the cuvette containing the nucleic acid sample or
standard suspended in the same solution as the blank and take a reading.
It is important that the sample be suspended in the same solution as used for the blank.
Variations between cuvettes (typically minimal) can be checked by measuring both
cuvettes with blank solution. Any additional background can be subtracted.
4. Repeat this process at 280, 260, and 230 nm for each sample before moving on to the
next.

For dual-beam spectrophotometers


1. Pipette 1.0 ml of 1× TE buffer/MQ water into two quartz cuvettes.
2. Place the cuvettes in a dual-beam spectrophotometer, read at 325 nm and zero the
instrument.
Use this blank solution as the reference.
3. Remove the cuvette from the sample position and replace the blank solution with the
nucleic acid sample suspended in the same solution as the blank. Insert the cuvette into
the spectrophotometer and take a reading.
4. Repeat readings at 280, 260, and 230 nm for each sample before moving on to the next.
It is important that the nucleic acid be suspended in the same solution as used for the
blank.
More sophisticated double-beam instruments will initially scan various wavelengths for
each sample, and this step may not be required.

OBSERVATIONS:
SAMPLE 230 260 280
OD

To determine the concentration of nucleic acid present, using the A260 values obtained:

Conc. = 1 x A260 x Dilution Factor

An A260 of 1.0 indicates


50 μg/ml dsDNA,
33 μg/ml ssDNA,
40 μg/ml ssRNA

For dsDNA or ssDNA and single-stranded RNA (ssRNA): These equations assume a 1-cm
path length spectrophotometer cuvette and neutral pH.
2
The calculations are based on the Lambert-Beer law, A = εcl, where A is the absorbance at a
particular wavelength, c is the concentration of DNA, l is the path length of the
spectrophotometer cuvette (typically 1 cm), and ε is the extinction coefficient or molar
absorptivity.

To estimate the purity of the nucleic acid sample using the A260/A280 ratio and readings at
A230 and A325
• Ratios of 1.8 to 2.0 and 1.9 to 2.0 indicate highly purified preparations of DNA and
RNA, respectively.
• Contaminants that absorb at 280 nm (e.g., protein) will lower this ratio.
• Proteins in general have A280 readings considerably lower than nucleic acids on an
equivalent weight basis. Thus, even a small increase in the A280 relative to A260
(i.e., a lowering of the A260/A280 ratio) can indicate severe protein contamination.
• Other commonly used buffer components absorb strongly at 260 nm and can cause
interference if present in high enough concentrations. EDTA, for example, should not
be present at >10 mM.
• Absorbance at 230 nm reflects contamination of the sample by phenol or urea.
• Absorbance at 325 nm suggests contamination by particulates and dirty cuvettes.

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