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DNA Quantification by Spectrophotometer New

The document discusses DNA quantification using a spectrophotometer. [1] A spectrophotometer measures the concentration of a solute by transmitting light through a solution and measuring light absorption. [2] DNA absorbs UV light strongest at 260nm due to its structure, and an A260:A280 ratio of 1.8 indicates pure DNA. [3] To quantify DNA concentration, an optical density reading at 260nm of 1.0 corresponds to 50 micrograms of DNA per milliliter in a 1cm cuvette.

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123 views6 pages

DNA Quantification by Spectrophotometer New

The document discusses DNA quantification using a spectrophotometer. [1] A spectrophotometer measures the concentration of a solute by transmitting light through a solution and measuring light absorption. [2] DNA absorbs UV light strongest at 260nm due to its structure, and an A260:A280 ratio of 1.8 indicates pure DNA. [3] To quantify DNA concentration, an optical density reading at 260nm of 1.0 corresponds to 50 micrograms of DNA per milliliter in a 1cm cuvette.

Uploaded by

Nimra Zaigham
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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LAB 7:

DNA quantification
by
spectrophotometer
Q1. Principle of DNA quantification by spectrophotometer?

Spectrophotometers:

A spectrophotometer is a piece of laboratory equipment that transmits light through a


solution to determine the concentration of a target solute by measuring the intensity of
light detected.

Principle:

It operates on the basis of a simple principle in which spectrometer produces a desired


range of light wavelengths, then lens transmits a straight beam of light (photons) that
passes through a prism to split it into several component wavelengths ,then the slit
transmits only the desired wavelengths that passes through a sample solution in
cuvette. The photometer detects the amount of photons that is absorbed and displays
it.

Figure 1 Spectrophotometers:

Q2. What will be the result of DNA quantification by spectrophotometer if cuvette is inserted
with the opaque side in the spectrophotometer?

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Answer: The cuvette must be inserted with transparent side facing the
spectrophotometer beam so rotating the cuvette by 180 degrees can give a significantly
different reading.

The purpose of the opaque sides is to prevent light scattering out of the cuvette at the
sides for certain types of samples.

Q3. At which wavelength does DNA show absorbance?

DNA has absorbance maxima for UV light at 260 nm due to the resonance structure of
the purine and pyrimidine bases.

Q4. What is optical density (O.D) and its relationship with the amount (concentration) of
DNA to be determined?

Optical Density:

Answer: Optical Density is the optical attenuation per centimeter of material as


measured using a standard spectrophotometer, typically specified with a 1 cm path
length. It is defined as:

“The ratio of the intensity of light absorbed by a material to the intensity of light
transmitted.”

General relation with DNA concentration:

1.0 O.D 260nm = 50µg DNA

Q5. O.D of blank?

Answer:

Optical density of blank is 0.00.

By placing the blank, we want to calibrate the instrument so that it will ignore any light
absorbed by the solution. By adjusting the light control knob, it shows 100% light
transmitted, 0 light absorbed. In reality, some small fraction of the light is scattered or
absorbed by the blank, but by calibrating the instrument in this way you can ignore that.

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When you insert your sample, the instrument will report only the fraction of the light
that is absorbed.

Q6. How the purity of DNA sample is determined (what is A260:A280 ratio)?

A260:A280 ratio

The ratio of absorbance maximum i.e. 260nm to the absorbance at 280 nm is used as a
measure of purity in DNA extractions.

For pure DNA samples, the maximum absorbance occurs over a broad peak at around
260 nm; at 280 nm it only absorbs about half as much UV light compared to 260 nm.
Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 or lower
ratios indicate more contaminants are present.

Q7. For pure DNA sample what should be the value of A260:A280 ratio?

A 260/280 ratio of 1.8 generally accepted as “pure” for DNA.

Q8. Peaks at A230 and A280 correspond to what in the given testing sample?

A230 and A280 correspond to the wavelengths at which maximum possible impurities
will absorb.

Q9. What will be the O.D of the given sample (1ug/ml) at A260 and cuvette size (1cm)?

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Q10. Problem:

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