we are going to find out how spectrophotometry help to analyze the Concentration by 260nm light and then purity by 260/280 and 260/230 rations

1. SPECTROPHOTOMETERY
(NUCLEIC ACID
QUANTIFICATION)
BY ASFANDYAR

2. WHY WE NEED TO QUANTIFY?
• Reactions that use nucleic acids often require particular amounts and purity
for optimum performance.
• To check concentration and purity of DNA/RNA present in the solution and to
determine if samples are useful for downstream applications like: PCR and
restriction enzyme
• The process of determining the concentration of DNA or RNA and their
purity in a mixture is called Nucleic acid quantification.
• One of the more commonly used practices to quantify DNA or RNA is the use
of spectrophotometric analysis using a spectrophotometer

3. HOW WE QUANTIFY NUCLEIC ACID?
• Nucleic acids absorb ultraviolet (UV) light due
to the heterocyclic rings of the nucleotides;
the sugar-phosphate backbone does not
contribute to absorption.
• The wavelength of maximum absorption for
both DNA and RNA is 260nm
• The absorption properties of DNA can be used
for detection, quantification and assessment of
purity

4. SPECTROPHOTOMETER
• An instrument employed to measure the amount of light that a sample
absorbs.

5. MEASURE OF NUCLEIC ACID CONCENTRTION
• When monochromatic light (light of a specific wavelength) passes through a solution there is
usually a quantitative relationship (Beer's law) between the solute concentration and the
intensity of the transmitted light, that is, the more concentrated the specimen is, the less light
is transmitted through it.
• The concentration of a substance in directly proportional to the amount of light absorbed.

7. 1 OPTICAL DENSITY OF DNA
0.02 x 50= 11 x 50 = 50ug
1 0D@260nm =
50ug/ml

8. OPTICAL DENSITY OF NUCLEIC ACIDS
• 1 O.D. at 260 nm for double-stranded DNA = 50 ug/ml of dsDNA
• 1 O.D. at 260 nm for single-stranded DNA = 20-33 ug/ml of ssDNA
• 1 O.D. at 260 nm for RNA molecules = 40 ug/ml of RNA

9. MEASUREMENTS OF SPECTROPHOTOMETER
• Measures DNA, RNA (A260) and Proteins (A280) concentrations and sample purity (260:280).
Absorbance at 260 nm
Nucleic acids absorb UV light at 260 nm due to the aromatic base within their structure making it the
standard for quantitating nucleic acid samples.
Absorbance at 280 nm
The 280 nm absorbance is measured where proteins and phenolic compounds have a strong
absorbance. Similarly, the aromaticity of phenol groups of organic compounds absorbs strongly near
280 nm.
Absorbance at 230 nm
Many organic compounds have strong absorbances at around 225 nm. In addition to phenol, TRIzol,
and chaotropic salts, the peptide bonds in proteins absorb light between 200 and 230 nm.

10. PURITY
• Historically, the ratio of absorbances at these wavelengths has been used as a measure of
purity in both nucleic acid and protein extractions.
• A ratio 260/280 of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally
accepted as “pure” for RNA.
• Similarly, absorbance at 230 nm is accepted as being the result of other contamination;
therefore the ratio of 260/ 230 is frequently also calculated.
• Expected 260/230 values are commonly in the range of 2.0–2.2.

11. 260/280
• 260/280 Ratios Abnormal 260/280 ratios usually indicate that the sample is
either contaminated by protein or a reagent such as phenol or that there was
an issue with the measurement.
• A low A260/A280 ratio may be caused by:
• Residual phenol or other reagent associated with the extraction protocol
• A very low concentration (>10 ng/µL) of nucleic acid
• High 260/280 purity ratios are not indicative of an issue.

12. 260/230
• Some contaminants have characteristic profiles, e.g. phenol, however many contaminants present
similar characteristics: absorbance at 230 nm or less. Abnormal 260/230 values may indicate a problem
with the sample or with the extraction procedure, so it is important to consider both.