Laboratory Manual

Name: Momina Abdul Qadir

Class: BS 2ND year

Course Title: Molecular Biology

Course Code: BTH- 2052

Department of Biotechnology
Jinnah University for Women
5-C Nazimabad

1|Page
 Certificate

Certified that Miss _____________________________________

D/o _________________________________________________

of ______________class has carried out the necessary practical work as

prescribed by the Department of Biotechnology, Jinnah University for Women

for the year ___________

Date:_____________

Course Incharge

Head of the Department

2|Page
 Index
Sr. Date Experiment Pg. No Initial Remarks
No
Laboratory solution
1 15.Aug.19 4-7
preparations and numerical.
Extraction of DNA from
2 20.Aug.19 8-9
Onion
Extraction of DNA from
3 29.Aug.19 10-11
different samples.

4 7.Sep.19 12
Preparation of Agar plate.

Isolation of pure culture of

5 15.Sep.19 13-15
E.coli by streak plate method
Preparations of media
6 26.Sep.19 16
(nutrient broth)
To grow the culture of E.coli
7 5.Oct.19 17-18
in Nutrient Broth
Isolation of genomic DNA
8 12.Oct.19 20-21
from E.coli.
Analysis of DNA by
9 18.Oct.19 22-23
Spectrophotometer

To perform Gel
10 18.0ct.19 24-27
electrophoresis

3|Page
 Lab #1

OBJECT: laboratory solution preparations and numerical.

THEORY:

Stock Solution:
A stock solution is a concentrated solution that will be diluted to some lower concentration
for some actual use to save preparation time, conserve material, reduce storage space and
improve the accuracy with which working lower concentration solutions are prepared.
Formulae:
C1V1= C2V2
C1= initial concentration
C2= final concentration
V1= Initial volume
V2= final volume

Working Solution:
Working solution is a name given to a chemical solution usually made from diluting or
combining stock or standard solution

Solution:
Solution is a homogenous mixture composed of two or more substances. A solution have two
parts: a solute and a solvent.

Solute:
A solute is defined as the substance that is dissolved in a solution. For solution of fluid, the
solvent is present in greater amount then the solute.
For example: Usually, a solute is a solid that is dissolved into a liquid. An everyday example
of a solute is salt in water.

Solvent:
The component of a solution that is present in the great amount. It is the substance in which
the solute is dissolved.
For example: The solvent for sea water is water. The solvent for air is nitrogen.

4|Page
Numericals:

Q1) Prepare 100mL of 0.5M NaCL.

A1)Data:
Molar mass = 0.5M
Molecular weight = 58gm
Volume = 100 Ml
Amount =?
Solution:
Amount = molar mass * molecular weight * volume / 1000
Amount = 0.5 * 58 * 100/1000
Amount = 2.9g/100mL
Dissolve 2.9g of NaCL in small amount of distilled water. Then make up its volume up to
100mL.
Q2) Prepare 175mL of 0.2M NaCL.
A2) Data:
Molar mass = 0.2M
Molecular weight = 58gm
Volume = 175 Ml
Amount =?
Solution:
Amount = molar mass * molecular weight * volume / 1000
Amount = 0.2 * 58 * 175/1000
Amount = 2.03g/175mL
Dissolve 2.03g of NaCL in small amount of distilled water. Then make up its volume up to
175mL.

Q3) From a working solution Prepare 0.5M of NaCL. Make 200mL of 0.1M.
A3) Data:
Concentration of working solution = C2 = 0.2M
Volume of working solution = V2 = 200mL
Concentration of Stock solution = C1 = 0.2M
Volume of Stock solution = V1=??
Solution:
C1V1 = C2V2
V1 = 0.1 *200/ 0.5
V1 = 40mL + 160mL distilled water.
Q4) Prepare 250mL of 2% CaCL2.
A4) Solution:
2%of CaCL2 = 2/100
Volume = 250mL
g = 2/100 * 250
g= 5gm/ 250mL
Q5) Prepare 150mL of 3% NaCL Solution.
A5) Solution:
3% of NaCL = 3/100
Volume = 150mL
g = 3/100 * 150
5|Page
g= 4.5gm/ 150mL

Q6) Prepare 100mL of 0.1N HCL from standard stock solution.

A6) Data:
Concentration of working solution = C2 = 0.1M
Volume of working solution = V2 = 100mL
Concentration of Stock solution = C1 = 12M
Volume of Stock solution = V1 =??
Solution:
C1V1 = C2V2
V1 = 0.1 *100/ 12
V1 = 0.8 + 99.2mL.
Don’t add water in acid always add acid drop by drop. Take 99.2mL of distilled water in a
container and then add 0.8 mL HCL drop by drop.
Q7) Prepare 100mL of 0.1Mtris-HCL buffer ,pH 7.5.
A7) Data:
Molar mass = 0.1M
Molecular weight = 121.1gm
Volume = 100ml
Amount =?
Solution:
Amount = molar mass * molecular weight * volume / 1000
Amount = 0.1 * 121.1 * 100/1000
Amount = 1.21gm.

Dissolve 1.21g of tris in small amount of distilled water and place the flask on magnetic
stirrer and add HCL drop by drop until the desirable pH is attained. Then add distilled water
and make up its volume up to 100mL.

Q8) Prepare 350mL of 0.5M tris-HCL buffer ,pH 7.2.

A8) Data:
Molar mass = 0.5M
Molecular weight = 121.1gm
Volume = 350ml
Amount =?
Solution:
Amount = molar mass * molecular weight * volume / 1000
Amount = 0.5 * 121.1 * 350/1000
Amount = 21.19 gm.
Dissolve 21.19g of tris in small amount of distilled water and place the flask on magnetic
stirrer and add HCL drop by drop until the desirable pH is attained. Then add distilled water
and make up its volume up to 350mL.

Q9) Prepare 0.1M Tris-HCL buffer containing 25mM CaCL2 (pH = 8.0) in 100 mL.
A9) Solution:
Data:
Molar mass of Tris= 0.1M
Molar mass of CaCL2= 25mM = 0.025M

6|Page
Molecular weightTris = 121.1gm
Molecular weight Of CaCL2= 111gm
Volume = 100ml
Amount of tris and CaCL2 =?
Solution:
For Tris For CaCL2

Amount = molar mass * molecular weight Amount = molar mass * molecular weight
* volume / 1000 * volume / 1000
Amount = 0.1 * 121.1 * 100/1000 Amount = 0.025 * 111 * 100/1000
Amount = 1.211 gm. Amount = 0.277 gm.

Dissolve 1.211g of tris + 0.277gm of CaCL2 in small amount of distilled water and place the
flask on magnetic stirrer and add HCL drop by drop until the desirable pH is attained. Then
add distilled water and make up its volume up to 350mL.

Q10)Prepare 225M Tris-HCL buffer containing 1.75M CaCL2 (pH = 7.8) in 250 ml.
A10) Solution:
Data:
Molar mass of Tris= 255M
Molar mass of CaCL2= 1.75M
Molecular weightTris = 121.1gm
Molecular weight Of CaCL2= 111gm
Volume = 250ml
Amount of tris and CaCL2 =?
Solution:
For Tris For CaCL2

Amount = molar mass * molecular weight Amount = molar mass * molecular weight
* volume / 1000 * volume / 1000
Amount = 225 * 121.1 * 250/1000 Amount = 1.75 * 111 * 250/1000
Amount = 6811.87 gm. Amount = 48.56 gm.

Dissolve 6811.87g of tris + 4856.25gm of CaCL2 in small amount of distilled water and place
the flask on magnetic stirrer and add HCL drop by drop until the desirable pH is attained.
Then add distilled water and make up its volume up to250mL.

7|Page
 LAB#2

OBJECT: Extraction of DNA from Onion

Theory:

DNA:
Deoxyribonucleic Acid (DNA) is a molecule that is present in the cell in all living organism.
It contain the genetic information found in every living cell in the hereditary “blue print” of
life.

ONION: the onion Elliconceps is diploid (2n=16) plant with a haploid genome size of about
17pg + have 15 million base pairs.

Onion is used for DNA extraction because it has low starch context which allow DNA to be
seen clearly.

Role of Detergent + Salt:

The detergent cause the cell membrane to break down(cell lyses) by dissolving the lipids and
proteins of the cell distributing the bonds that hold the cell membrane together the detergent
then forms complexes with these lipids and proteins causing them to precipitate out of the
solution

The use of NaCl salt shields the negative phosphate end of the DNA which allows these ends
to come closer so can precipitate out of cold ethanol solution.

Role of Ethanol
The DNA released from the cell nucleus is dissolved in the water/detergent/specimen solution
and cannot be seen. DNA precipitates out of solution in ethanol, where it can be seen. In
other words, DNA is less soluble in ethanol than in water. When ethanol is added, the
separate DNA molecules clump together and become visible.

PROCEDRE:

Wear gloves & do not touch the inside of the container because DNA Are enzyme from the
hand will break the DNA into Small fragment.

1. Wearing plastic gloves, dice a medium size onion no length than 3mm

2. Weight out 6 gm. of onion of 100ml homogenizing medium to the dice onion & incubate
the beaker in a 52 degree C water bath for 15 min. this heat treatment soften the onion tissues
& allow presentation of homogenizing solution. It also donate used many enzymes that could
interface with the isolation procedure.

3. Quickly cool your procedure from 10 to 14 degree in an ice bath for 6 min.

4. Filter the homogenate into a chilled beaker.

8|Page
5. Measure out 80 ml of ice paused ethanol and slowly add the ethanol down the side of the
beaker until the white DNA participate appears.

6. Spout out the DNA with the help of glass rod & place in 50% ethanol.

RESULT:

DNA from onion have been extracted and stored for further use.

Extraction of DNA from onion

9|Page
 LAB#3

OBJECT: Extraction of DNA from different samples.

Theory:

DNA:
Vegetables, fruits, cereals and meat all plant and animal products contain DNA in various
amounts. DNA, the carrier of genetic information, the recipes or blueprints of an organism, is
made up of four different chemical units. These can be digested and used just like any other
nutrient in our food. A pound of broccoli, for example, contains about a tenth of an ounce
DNA.

Usually DNA is degraded during cooking, but even if DNA is eaten uncooked, like in the
case of apples, a tomato, or a salad, the DNA is degraded rapidly in our stomach. Eating
DNA should not be a concern: even mother’s milk contains high amounts of DNA, because
newborns have a higher need for them.

DNA is found in the vast majority of an organism’s cells. This molecule functions in
providing the plans for building the cell’s proteins. Proteins are vital to the cell, functioning
as structural components as well and functional molecules of the cell. Enzymes and
hormones, for example, are primarily proteins. DNA is located in three different structures
within eukaryotic cells. Nuclear DNA is located in the nucleus; mitochondrial DNA is
located in mitochondria, and; chloroplast DNA is located in the chloroplast of eukaryotic
photosynthetic cells. By far, the greatest amount of DNA is located in the nucleus of
eukaryotic cells. In this laboratory investigation, you will attempt to extract DNA from two
different organisms using slightly different methods.

Role of Detergent + Salt:

The detergent cause the cell membrane to break down(cell lysis) by dissolving the lipids and
proteins of the cell distributing the bonds that hold the cell membrane together the detergent
then forms complexes with these lipids and proteins causing them to precipitate out of the
solution

The use of NaCl salt shields the negative phosphate end of the DNA which allows these ends
to come closer so can precipitate out of cold ethanol solution.

Role of Ethanol
The DNA released from the cell nucleus is dissolved in the water/detergent/specimen solution
and cannot be seen. DNA precipitates out of solution in ethanol, where it can be seen. In
other words, DNA is less soluble in ethanol than in water. When ethanol is added, the
separate DNA molecules clump together and become visible.

Materials:

 Given Samples (tomato, Onion and apple)

 Ethanol

10 | P a g e
  Salt
 Water
 Beaker
 Filter paper
 Test tubes
 Spatula
 Funnel

PROCEDRE:

1. Chop the samples into very fine pieces. Mix 2 table spoons of salt with 1/2 teaspoon of
dishwashing detergent (or DIAL, liquid soap) in a glass.
2. Add the chopped sample into it and mis with spatula.
3. Strain the well-blended mixture into separate test tubes through filter paper.
4. Carefully add cold alcohol (95% ethanol). The alcohol should float on top of the
mixtures, but don’t worry if it mixes a bit.
5. At the layer between the Mixtures and ethanol you will see a whitish, "snotty-looking",
substance. This is DNA. Carefully swirl the solutions in the glass, to get more DNA.

RESULT:

DNA from various samples have been extracted.

APPLE TOMATO ONION

White cloudy structure and was Intermediate between apple Blurred precipitates and
much clear then the rest. and onion. most unclear.

Extraction of DNA from Apple

11 | P a g e
 LAB#4

OBJECT: Preparation of Agar plate.

Materials and Equipments

 Distilled Water
 Measuring Cylinder
 Cotton
 Gloves
 Sterile, Empty Petri Dishes
 Bunsen Burner
 Autoclave
 Incubator
 Sterile Media

Procedure

1. Prepare a medium as in previous EXERCISE.

2. Cool the sterilized medium to 55°C.
3. Take out the cotton plug and flame the mouth of
the flask over a Bunsen burner and then pour the
medium into sterile, empty Petri dishes(15-20mL
into each Petri dish).
4. Keep the Petri dishes horizontally until the
medium completely solidifies.
5. Turn dishes upside-down and stack them up for
storage.
6. Label the plates according to the type of the medium.
7. In case of longer storage, Petri plates must be placed into plastic bags or boxes to avoid
drying out.

12 | P a g e
 LAB#5

OBJECT: Isolation of pure culture of E.coli by streak plate method

Theory:

Nutrient Agar:
Nutrient Agar is a general purpose, nutrient medium used for the cultivation of wide variety
or range of non – fastidious organisms. It is typically contain 0.5 % peptone, 0.3 % beef/yeast
extract, 1.5 % agar, 0.5% sodium chloride. pH adjusted to 7.4.

Four Way Streak Method:

The streak plate method is a rapid qualitative isolation method. The techniques commonly
used for isolation of discrete colonies initially require that the number of organisms in the
inoculums be reduced. It is essentially a dilution technique that involves spreading a loopful
of culture over the surface of an agar plate. The resulting diminution of the population size
ensures that, following inoculation, individual cells will be sufficiently far apart on the
surface of the agar medium to effect a separation of the different species present. Although
many type of procedures are performed, the four ways or quadrant streak is mostly done.

Materials:

 Nutrient Agar
 Spatula
 Weighing balance
 Distilled water
 Measuring cylinder
 Conical flask
 Magnetic stirrer
 Petri plates
 Autoclave
 Mixed culture of E.coli

CALCULATION:

For Nutrient Agar:

28 𝑔𝑚
Standard = 1000𝑚𝑙
28×50 1.4𝑔𝑚
For 50ml = =
1000 50𝑚𝑙

For Agar-Agar:
Agar Agar 1% in 100ml
1×50 0.5 𝑔𝑚
For 50ml= 100 = 100𝑚𝑙

13 | P a g e
PROCEDURE:

Preparation of nutrient agar:

 Weigh 1.4 gm of nutrient agar powder.
 Weigh 0.5 gm of agar-agar.
 Dissolved nutrient agar powder and agar-agar in 50 ml of distilled water in conical
flask.
 Place the flask on magnetic stirrer for complete dissolution.
 Autoclave the dissolved mixture at 121°C for 15 minutes.
 Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
 Pour nutrient agar into each plate and leave plates on the sterile surface until the agar
has solidified.
 Store the plates for further use.

Four Quadrant Streak:

1. Hold an inoculation loop in your right hand.
2. Flame the loop red hot it and allow it to cool.
3. Lift the lid of the small tube containing the inoculum with your left hand.
4. Insert the loop into the culture broth and withdraw. At all times hold the loop as still
as possible.
5. Replace the cap of the test tube using the little finger of your right hand. Place the
small tube into rack.
6. Partially lift the lid of the Petri dish containing the solid medium.
7. Place a loopful of the culture on the agar surface on the area 1. Flame the loop and
cool it for 5 seconds by touching an unused part of the agar surface close to the
periphery of the plate, and then drag it rapidly several times across the surface of
area1.
8. Remove the loop and close the Petri dish.
9. Reflame and cool the loop, and turn the Petri dish 90°C then touch the loop to a
corner of the culture in area1 and drag it several times across the agar in area 2, hitting
the original streak a few times. The loop should never enter area 1 again.
10. Remove the loop and close the Petri dish.
11. Reflame and cool the loop and again turn the dish 90°C anticlockwise. Streak area 3
in the same manner as area 2, hitting last area several times.
12. Remove the loop and close the Petri dish.
13. Flame the loop, again turn the dish 90°C and then drag the culture from a corner of a
area3 across area 4, contacting area 3 several times and drag out the culture. Using a
wider streak. Do not let the loop touch any of the previously streaked areas.
14. Remove the loop and close the Petri dish.
 Tape the plate closed and incubates the plate in an inverted position in an incubator
for 24-48 hours.

14 | P a g e
RESULT:

The colonies of E.coli appeared on the agar plate after 24 hrs. The pure culture of E.coli has
been isolated which will be used in the next experiment to observe the growth pattern in
nutrient broth. The colony was off-white in color. Each colony was slightly raised and had
entirely fixed margin.

Colonies of E.coli observed after 24 hours

15 | P a g e
 LAB#6

OBJECT: Preparations of media (nutrient broth)

THEORY

This medium is a standard medium for a broad range of bacteria. With the addition of blood,
ascites fluid or serum they can also be used to cultivate streptococci, pneumococci and
erysipelas species etc. Peptone and yeast extract provide nitrogenous compounds, vitamin B
complex, amino acids and other essential growth nutrients. Sodium chloride is used to
maintain the osmotic balance and glucose is the fermentable carbohydrate source of the
media.

MATERIALS AND EQUIPMENTS

 Nutrient broth
 Spatula
 Measuring cylinder
 Flask
 Cotton plugs
 Weighing balance
 Glass rod
 Autoclave

Calculation of Nutrient Broth

 Standard concentration: 13/1000ml

13
 For 100 ml : 100 × 100 = 1.3grams/100 ml of distilled water.

PROCEDURE

1. Weigh out 1.3 g of Nutrient broth and dissolve in 100 ml distilled water in a flask.
2. Manually mix the nutrient broth in distilled water and cover with cotton plug.
3. Autoclave for 15 – 20 minutes at 121°C.
4. Store until next lab.

16 | P a g e
 Lab # 7

OBJECT: To grow the culture of E.coli in Nutrient Broth

THEORY:

Nutrient Broth:

Nutrient broth is a liquid medium which is prepared to grow a wide variety of bacteria in the
labs. The composition of the nutrient broth is the same as the nutrient agar except for the
addition of agar. Agar is not used since it is a solidifying agent.

Peptone and yeast extract provide nitrogenous compounds, vitamin B complex, amino acids
and other essential growth nutrients. Sodium chloride is used to maintain the osmotic balance
and glucose is the fermentable carbohydrate source of the media.

MATERIALS
 Nutrient broth
 Spatula
 Measuring cylinder
 Distilled water
 Flask
 Cotton plugs
 Weighing balance
 Glass rod
 Autoclave
 Culture containing Petri plate

Calculation:
13𝑔𝑚
Standard = 1000𝑚𝑙

13×150 1.95 𝑔𝑚
For 150 ml = = 150𝑚𝑙
1000

PROCEDURE:

a. Preparation of nutrient broth:

1. Weigh 1.95gm of nutrient broth.
2. Dissolved nutrient broth powder in 150 ml of distilled water.
3. Mix well and dissolve with the help of magnetic stirrer.
4. When it’s perfectly dissolve pour this in test tubes.
5. Cover all the test tubes and sterilize in autoclave at 121°C for 15 minutes.
6. After autoclave take out the culture containing petri plates, put on the burner, clean
the bench top with ethanol.
7. Sterile the wire loop in a flame till it become red hot and then cool it, open the Petri
plate containing culture near the flame and with the help of wire loop take the culture
from the plate.

17 | P a g e
 8. Now open the tube containing nutrient broth and inoculate the culture in the tube.
9. Gently close the tube and incubate the tube at 37 °C for 24 hrs.
10. After 24 hr observe the result

RESULT:

E.coli is highly motile and showed turbidity throughout the tube after 24 hrs. This cultural
growth can be used in future experiment to determine the optical density. For this purpose,
spectrophotometer is used. In addition to oxygen requirements, there are other factors that
can influence microbial growth patterns in liquid. Motile bacteria (those with flagella) can
swim. Their movement will create a uniform cloudiness (turbidity) in the broth. Although
there are many advantages to growing bacteria in broth, one of the main disadvantages is that
bacterial colonies do not form in a liquid suspension.

Turbid growth was observed after

24 hrs.

18 | P a g e
 Lab # 8

OBJECT: Isolation of genomic DNA from E.coli.

Theory:

E.coli is a gram negative facultative anaerobic (that makes ATP by aerobic respiration if
oxygen is present, but is capable of switching to fermentation or anaerobuic respiration, if
oxygen is absent) and known sporalating bacteriuym.

Optimum growth of E.coli occurs at 37 degree Celsius (98.6 F), but some laboratory strains
can multiply 8 temperatures upto 49 degree Celsius (120 F). E.coli grows in a variety of
defined laboratory media, such as lysogeny broth, or any medium that contains glucose,
ammonium phosphate monoibasic, sodium chloride, magnesium sulfate, potassium phosphate
dibasic, and water. Growth can be driven by aerobic or anaerobic respiration.

E.coli is classified as a facultative anaerobe. It uses oxygen when it is present and available.
It can however, continue to growth in the absence of oxygen using fermentation or anaerobic
respiration.

E.coli on agar form circular colonies and are translucent.

Types of DNS found in Bacterial E. coli

 Plasmid DNA
 Chromosomal genomic DNA

Material:

 Juster
 Microcentrifuge
 Lysis solution
 70% ethanol
 Burner
 Matchstick
 Cotton
 Micropipette

Procedure:

1. Centrifuge at 5000 rpm for 10 minutes to pallet the cell.

2. Transfer the supernatant back into the culture growth
3. Resuspend the cell pallet in 500 mL of lysis solution(weight of 0.2 M NaOH and
1% SDS) with the help of micropipette
4. Incubate at 80 degree Celsius for 5 minutes to completely lysis the cell
5. Cool the contents in centrifuge at 5000 rpm for 10 minutes’
6. Transfer the supernatant containing 600 micro Liters of isoprpynol
7. Mix the solution by invert mixing for 15 times
19 | P a g e
 8. Centrifuge it at 5000 rpm for 5 minutes at 6000 micro liters of 70% ethanol and
invert the tube several times to wash the DNA pallet
9. Centrifuge it at 5000 rpm for 10 minutes
10. Carefully pour ethanol supernatant and invert the tube on clean tissue paper
11. Allow the pallet to dry for 10 to 15 minutes
12. Store at 0-20 degree Celsius until use

Result:

The genomic DNA of E.coli has been isolated.

DNA of E.coli in micro centrifuge

tube

20 | P a g e
 Lab #9

OBJECT: Analysis of DNA by Spectrophotometer

THEORY

A spectrophotometer is employed to measure the amount of light that a sample absorbs. The
instrument operates by passing a beam of light through a sample and measuring the intensity
of light reaching a detector.

It operates on Beer’s law:

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.

Absorption spectrophotometry is a widely used technique based on the property of molecules

to absorb light at specific wavelengths. The amount of DNA in a solution can be determined
using spectrophotometer.

Quantification of nucleic acids is commonly used in molecular Biology to determine the

concentration of DNA and RNA present in the mixture. Both DNA and RNA exhibit strong
absorbance of UV due to the presence of conjugated double bonds of the constant purine and
pyramidine bases and these have characteristics of OD (optical density) of absorbance
maximum at 260nm which is linearly related with the concentration of the DNA in the
solution up to the OD value of 2.

Spectrophotometric Measures

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 moieties within their
structure. Purine (thymine, cytosine and uracil) and pyramidine (adenine and guanine) both
have peak absorbance at 260 nm, thus 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.

21 | P a g e
MATERIALS REQUIRED

 Buffer
 Quarts cuvette
 Distilled water
 Tissue paper
 Conical flask
 Beaker
 UV spectrophotometer
 DNA Sample

PROCEDURE:

1. Turn on the spectrophotometer. Check the display is on and ready. Turn on the lamp
source and allow it to warm.
2. Select the absorbance reading mode. Enter the wavelength i.e. 260nm.
3. Rinse the cuvette with distilled water and clean it with tissue paper.
4. First take the blank reading by placing the buffer in cuvette and press read.
5. Now remove the buffer from cuvette and take the DNA sample in a cuvette and take it
absorbance at 260nm.
6. Follow the same procedure for taking the absorbance at wavelength 280nm.

OBSERVATION:

260nm 280nm A260/A280

0.206 0.177 0.206/0.177 = 0.16
CALCULATION:

1OD260 unit=50μg/ml DNA

Abs1͢→50 μg/ml

0.206 absorbance means 50/1 × 0.206

= 10.3µg/ml DNA

1OD280 unit=1mg/ml Protein

0.177 OD means 1/1 × 0.177

= 0.177mg/ml protein present

22 | P a g e
RESULT:

The absorbance at 260nm was 0.206. This shows that 10.3µg/ml of DNA is present. The
absorbance at 280nm was 0.177. This shows that 0.177mg/ml of protein is present. Pure
DNA preparations have an A260/A280 ratio of greater than or equal to 1.8. But here the ratio
of A260/A280 is less than 1.8. Therefore, it indicates the presence of proteins associated with
the DNA.

Several factors can influence the accuracy of the A260/A280 ratio. For example, readings
from very dilute samples will have very little difference between the absorbance at 260 and
280nm leading to inaccurate ratios. The type(s) of protein present will also have an effect.
Absorbance in the UV range by proteins is primarily the result of aromatic ring structures.
Proteins are composed of 22 different amino acids of which only three contain aromatic side
chains. Thus the amino acid sequence of proteins would be expected to influence the ability
of a protein to absorb light at 280 nm.

WORKING OF SPECTROPHOTOMETER

23 | P a g e