Molecular Biology Topics

QUANTITATIVE ANALYSIS OF DNA

Written by MicroDok

The quantitative analysis DNA is explained in this section

Aim of the experiment: To determine the amount, concentration and purity of the given DNA sample.

Principle: This experiment is purely an application of the Beer Lamberts’ Law which
states that the concentration of the sample is directly proportional to the absorbance of
light done by the sample. It is given by following expression:

A = Ԑ x C x L

The device UV spectrophotometer works on this principle and used to find the
concentration of the sample.
Concentration and quality of a sample of DNA is measured with a UV
spectrophotometer.
A standard graph can be drawn using different concentrations of DNA and OD (optical
density
) values.

MATERIALS REQUIRED FOR QUANTITATIVE ANALYSIS OF DNA

  • DNA sample
  • Tris EDTA (TE) buffer
  • UV spectrophotometer

Illustration of quantitative DNA analysis

PROCEDURE FOR QUANTITATIVE ANALYSIS OF DNA

  1. Take the DNA sample (10 ul) in TE buffer.
  2. Now dilute the above sample by the factor of 100 i. e, by taking 10µl
    of the sample in 990µl of TE buffer.
  3. After doing this take the optical density value at A260 & A280 and
    calculate the amount of DNA recovered.
  4. Use the following formula to determine the concentration of DNA:
    Total DNA (ug) = (A260) (50 ug/ml/A260) (100) (0.1 ml)

where 100 is the dilution factor and 0.1 ml is the total volume of the
DNA;

Quality: DNA quality measurement is based on the fact that OD at 260
nm is twice that at 280 nm if the solution contains pure DNA. If there is a
contaminant, there is some additional OD, which decreases the OD ratio
between 260 and 280 nm.
Clean DNA has a OD260/OD280 between 1.8 and 2.0

The NanoDrop can also be used for quantifying nucleic acid samples (DNA or RNA)

Illustration of the NanoDrop Lite

Results and Discussion:

 

References:  DEPARTMENT OF BIOTECHNOLOGY, SCHOOL OF BIOENGINEERING, SRM UNIVERSITY

 

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MicroDok

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