1.

In order for a photon of radiation to be absorbed by a substance, the energy content of the photons
must match the difference in energy between two different energy levels in the molecule. 

True

2. A chemical structure in a substance that absorbs light

CHROMOPHORE

3. Photons of light at the red end of the visible spectrum have a higher energy content than those at the
blue end. 

False 

4. linear distance between two successive amplitude maxima on the beam.

WAVELENGTH

5. number of oscillations of the wave per second the number of successive maxima passing a point per
second.

FREQUENCY

6. IS THE DISTANCE BETWEEN TWO SUCCESSIVE PEAKS AND IT IS EXPRESSED IN TERMS OF

NANOMETER.

c. Infrared

  a. Frequency

  e. All of the above

  d. Energy

  b. Wavelength

7. Fraction of light that passes through the sample

TRANSMITTANCE

8. Which of the following lamps provides a continuous spectrum of radiant energy in the visible, near
infrared and near ultraviolet regions of the spectrum?

TUNGSTEN-FILAMENT

 
9. Which of the following may be associated with reflectance spectrophotometry as it relates to the dry
reagent slide technique.

c. Unabsorbed, reflected light detected by photodetector.

10. In spectrophotometric analysis, what is the purpose of the reagent blank?

Correction for protein

   Correction color contribution of the reagents

11. 710 nm can be interpreted as:

E. Both A and C

    

12. Filters produce monochromatic light based on the principle of constructive interference of waves -
light waves enter one side of the filter and are reflected at the second surface. Filters pass a wide band
of radiant energy and have a high transmittance of the selected wavelength. Filters are made by placing
semi-transparent silver films on both sides of a dielectric such as Magnesium chloride.

C. 1st sentence is correct, 2nd sentence is incorrect and 3rd sentence is incorrect 

13. Monochromator provides polychromatic light and must generate sufficient radiant energy or power to
measure the analyte of interest. Prisms can be rotated, allowing only the desired wavelengths to pass
through an exit slit. A narrow light focused on a prism is refracted as it enters the more dense glass. 

A. 1st sentence is incorrect, 2nd sentence is correct and 3rd sentence is correct.

  

14. A Slight error in wavelength adjustments can introduce a  significant error in absorbance readings.
The higher The wave frequency, THE LONGER THE WAVELENGTH. Wavelength accuracy is the
wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator.

E. 1st sentence is correct, 2nd sentence is incorrect and 3rd sentence is correct.  

15. Characteristics of Light source except:

  Exceeds Spectral distribution range

16. Alkaline solutions should not be left standing in cuvets for prolonged periods because alkali slowly
dissolves glass producing etching is applicable to:

   C. Cuvettes

17. The function and consideration in using the cuvettes is/are:

  D. Cuvets with scratches on their optical surface scatter light and should be used.

  B. carefully hold the opaque side.

  E. Both A and C

  F. Both B and D

  C. Borosilicate glass and Soft glass can be used.

  A. sample is being placed.

18. Unwanted lights in spectrophotometer causes absorbance error. Unwanted lights limits the
maximum absorbance that spectrophotometer can achieve and is the most common cause of loss of
linearity at high-analyte concentration. The exit slit duty is to minimizes unwanted or stray light and
prevents the entrance of scattered light into the monochromator system.

  C. 1st sentence is correct, 2nd sentence is correct and sentence is incorrect.

19. The monochromator’s Filter is/are:

  E. Both A and D

  F. None of the above

  D. uses silver films

  A. usually pass a wide band of radiant energy and have a low transmittance of the selected
wavelength

  B. made of parallel grooves

  C. better resolution that prisms

20. It detects the amount of light that passes through the sample in the cuvet.

  B. Photodetector

21. Beer’s Law - concentration of the unknown substance is inversely proportional to the absorbed light
(absorbance or optical density) and directly proportional to the amount of transmitted light/reflected light
(% Transmittance).

  False

22. An alternate form of a gene or a gene variant

  B. Allele

23. Arrange the following sequence of spectrophotometer starting from the Light source

  D. Entrance slit, Monochromator, Exit slit, cuvette, photodetector, readout device

24. These tubes are limited to measuring low power radiation because intense light causes irreversible
damage to the photoelectric surface. This photodetector is:

  C. Photomultiplier tube

25. Sample blank  measures absorbance of the sample and reagent in the absence of endproduct, and
corrects the measurement for optical interference (like hgb) absorbing the wavelength of measurement.

  True 
26. A sequence of DNA that has a known function such as encoding proteins or controlling gene
expression.

  B. Gene

27. . Organic Isolation of DNA Extraction: The purpose of acidification by the use of acetic acid in DNA
extraction is to:

  A. precipitate the DNA

  C. for cell lysis

  B. to have a cell debris

  D. to isolate the liquid portion of the specimen

28. The purification is performed to avoid contaminations with other intracellular components

  True

29. Isolation of DNA: Solid Phase Isolation requires:

  DNA Absorption with Low Ph

30. Isolation of DNA can be used as a method:

  Solid-Phase Isolation

  crude lysis

  Inorganic Isolation Methods

  Organic Isolation Methods 

Introduction: 

Quantification and assessment of DNA/RNA and Protein purity and concentration, is first entry step in
most of molecular biology protocol routinely employed in many lab. DNA Quantitation is commonly
performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their
purity. There are two main approaches used by scientists to quantitate, or establish the concentration, of
nucleic acids (such as DNA or RNA) in a solution. These are spectrophotometric quantification and UV
fluorescence tagging in presence of a DNA dye.

Learning Objectives:

At the end of the topic, the students should be able to:

1. To recognize the importance of DNA Sample preparation.

2. Relate the accuracy of wavelength measurement for purified DNA sample.
3. To interpret the different wavelength for the isolation of purified DNA.

Principle of the Test:   

Spectrophotometric analysis is based on the principles that nucleic acids absorb ultraviolet light in a
specific pattern. In the case of DNA and RNA, a sample is exposed to ultraviolet light at a wavelength of
260 nanometers (nm) and a photo-detector measures the light that passes through the sample. Some of
the ultraviolet light will pass through and some will be absorbed by the DNA / RNA. The more light
absorbed by the sample, the higher the nucleic acid concentration in the sample.

Checking  quality  of  sample  for  detection  of  Protein,  or  other  contaminant  with DNA/RNA sample
is most common practice. For standard practice for DNA sample usually A260/A280 are used, as if ratio
is 1.8 indicate good pure quality of DNA, while ratio of 2.0 indicate contamination of RNA in sample and
same ratio less than or 0.6 is indicating protein in sample.

Materials: 

1. DNA/RNA Sample
2. Buffer

Procedure:

1. Start on spectrophotometer and adjust it to wavelength 260 nM.

2. Auto-zero spectrum with blank of TE buffer for DNA/RNA or  PBS buffer Protein sample. 
3. Withdraw buffer and rinse cuvette once with distilled water.
4. Fill cuvette with max volume with DNA/RNA/Protein sample.
5. Run absorbance spectrum in range of 230 to 330 Nm.
6. Run Peak value and find point with maximum absorbance in range of scan and note it
7. Further, take absorbance at 230, 260 and 280 Nm and note it.
8. Check quality of sample by taking ratio of A230/260/280, A260/280 and A260/230.
9. Calculate quantity of sample as per equation and note it.

Results of the test:

 Wavelength
Characteristic nature Remarks
(Nm)

Minimum  absorbance for  Measurements  are  generally  not  performed  at  this wavelength 
nucleic  acids. Peptide  because  commonly  used  buffers  and solvents,  such  as  Tris, 
215-230
bonds  in proteins absorb also  absorb  at  these wavelengths.  Contaminant  phenolate  ion, 
light.   thiocynate and other organic compound absorb at this range.

Purines absorbance maximum is  slightly below 260; pyrimidines

maximum, is slightly above 260. Purines have  a  higher  molar 
Nucleic  acid  have
absorptivity  than  pyrimidines.
260 maximum  absorbance at
this wavelength Therefore, the absorbance maximum and absorptivity of  a 
segment  of  DNA  depends  on  its  base composition.

270 Phenol  absorbs strongly Phenol may be contamination

280 Protein  absorbance peak Aromatic amino acid strongly absorb at 280 Nm.

Non  protein  and Nucleic 

330 or higher Presence of particulate matter causing light scattering
acid absorbance range

[TRANS] M3: DNA QUANTITATION FLUOROMETRY/FLUORESCENT SPECTROSCOPY

•Measures fluorescence related to DNA concentration in association with

SPECTROPHOTOMETRIC ANALYSIS OF DNA DNA-specific fluorescent dyes•Early method use DABA
•Modern method use DNA specific dye (Hoechst 33258)
•Principle: Nucleic acids absorb ultraviolet light in aspecific pattern. •PicoGreen=more sensitive
•Nucleic acids absorb light at 260 nm •OliGreen= single stranded DNA
•Absorptivity constants= (50 for DNA, 40 for RNA) •SyBrGreen II= RNA stain
•Concentration to Absorbance:
ELECTROPHORESIS
•Movement of molecules by an electric current
•Done in a matrix to limit migration and contain the migrating material
•Protein and Nucleic acids

•One optical density unit (or absorbance unit) at 260 nm =50 mg/L (or 50
g/mL) of DNA and 40 g/mL of RNA
GEL SYSTEMS
•Example 1:
A DNA preparation diluted 1:100 yields an absorbance reading of •Provide resistance to the movement of molecules
0.200 at 260 nm. To obtain the concentration in μg/mL, multiply: •Prevent diffusion and reduce convection currents
0.200 absorbance units x 50 μg/mL per absorbance unit x100= 1000 •Serve as a support medium
μg/mL •Agarosegels
If in this case, the DNA was eluted or resuspendedin a volume of •Polyacrylamide gel
0.5mL,the yield would be
:1000μg/mL x 0.5 mL= 500μg AGAROSE GELS
•Example 2: •Extracted from seaweed
An RNA preparation diluted 1:10 yields an absorbance reading of •Component of agar
0.500 at 260 nm. The concentrations is: •Linear polymer of agarobiose
0.500 absorbance units x 40 μg/mL per absorbance unit x10= 200
μg/mLIf in this case, the RNA was eluted or resuspendedin a volume
of 0.2 mL,theyield would be:
200μg/mL x 0.2 mL= 40μg
 BUFFER SYSTEMS

•To carry the current and protect the samples during electrophoresis
•Solution of a weak acid and its conjugate base
•Commonly used:
•Trisborate EDTA (TBE)
•Trisphosphate EDTA (TPE)
•Trisacetate EDTA (TAE)
•Advantages and disadvantages:
•TBE has a greater buffering capacity than TAE
•TBE is not recommended for some post-electrophoretic
isolation procedures.