NAME :- HARI OM SHARAN

ROLL NO :- AE-054
MOLECULAR BIOLOGY FILE
Submitted to Dr. Rashmi Ma’am

TABLE OF CONTENT
● Introduction to Molecular Biology.

● Preparation of LB media and inoculation of E.coli.

● Experiment 1:
Isolation of Plasmid DNA from E. coli
Isolation of Genomic DNA from E. coli
Quantification Using Agarose Gel Electrophoresis.

● Experiment 2: Isolation of Genomic DNA from Plant Samples Using CTAB Method.

● Experiment 3: Quantification of DNA Using Diphenylamine Reagent (Colorimetry).

● Experiment 5: Control of Translation in E. coli Using Antibiotic Inhibitors (Kanamycin).

 Introduction to Molecular Biology

Molecular biology is a sub-branch of science that deals with the structure, function, and interaction of
biological molecules. It mainly focuses on DNA, RNA, and proteins, which are the primary materials
found in living organisms. This branch of science gained momentum in the mid-20th century with the
revelation of the structure of DNA and has since caused a revolution in our knowledge of genetics, cell
biology, and biotechnology.

Thus, the central focus of molecular biology is to understand how genetic information encoded in DNA is
transcribed into RNA and translated into proteins. Since all cells' constituent functions are performed
primarily through proteins, this process applies uniformly in living organisms.

Molecular biological techniques, such as DNA extraction, DNA Quantification, gel electrophoresis, and
gene cloning, are used to experiment with genetic materials. These will allow scientists to "play" with
genes, proteins, and other molecules in great detail.

Molecular biology applications are unlimited, ranging from medicine, agriculture, and forensics to
biotechnology. For example, the molecular basis of many diseases is used as the basis for targeted
therapies and vaccines. Similarly, advancements in genetic engineering and cloning have presented new
avenues in biotechnology, such as genetically modified crops and gene therapy.

In this laboratory manual, we have several important techniques of molecular biology, including DNA
extraction, quantification, and analysis, as well as understanding genetic regulation and growth patterns.
Through these experiments, we would acquire better insights into the processes controlling life at the
molecular level.

[1]
Preparation of LB Media and Inoculation of E. coli

Objective:

To prepare LB (Luria-Bertani) media and inoculate E. coli bacteria for growth and culture.

Materials:

For LB Broth:

● Tryptone (10 g): A source of amino acids and peptides, essential for bacterial growth.
● Yeast Extract (5 g): A source of vitamins, growth factors, and minerals required for bacterial
growth.
● Sodium Chloride (NaCl) (10 g): Provides the necessary ionic strength for bacterial cells to
survive and maintain osmotic balance.
● Distilled Water (1 liter): To dissolve the above ingredients.
● pH Adjustment: pH should be adjusted to 7.0 -8.0 using NaOH .(if low)

Equipment:

● Autoclave (for sterilization)

● Glassware (flasks, beakers)
● pH meter or pH paper
● Stir bar and magnetic stirrer
● Sterile pipette or inoculation loop
● Incubator set at 37°C
● Shaking incubator

[2]
Procedure:

1. Preparation of LB Broth:

1. Weighed and Mixed Components:

 Weighed 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl.
 Added these to 800 milliliters of distilled water.

2. Dissolved the Components:

 Stirred the solution using a magnetic stirrer until all solids were dissolved.

3. pH Adjustment:
 Checked the pH of the solution using a pH meter or pH paper.
 Adjusted the pH to 7.0 using NaOH, if necessary.

4. Sterilization:
 Made the volume up to 1000 ml and sterilized the prepared media by autoclaving at 121°C for
15-20 minutes. This killed any potential contaminants and sterilized the medium for bacterial
growth.

5. Cooled the Media:

After autoclaving, allowed the LB broth to cool to room temperature in a sterile environment.

[3]
2. Inoculation of E. coli:

1. Prepare a Culture:
○ For inoculation, we use a fresh colony of E. coli from an agar plate or a stock E.coli.
culture.
○ Use a sterile inoculating loop to pick a colony or a small volume (1-2 µL) of the E. coli
culture from a stock or previous culture.
2. Inoculate the LB Broth:
○ Transfer the picked colony or liquid culture directly into the cooled LB broth.
○ Typically, a single colony or a small amount (1–2 µL) is enough to inoculate 5–10 mL of
LB broth.
3. Incubation:
○ We Place the inoculated flask in a shaking incubator set to 37°C and 200–250 rpm for
10-12 hours.

Observation:

● Inoculation into Broth:

○ After 12–18 hours of incubation, the broth should become turbid or cloudy, indicating
bacterial growth.
○ The degree of turbidity will increase as the bacteria multiply and reach higher densities.
● Inoculation into Agar
○ After incubating an LB agar plate, we should observe well-separated colonies of E. coli
that are typically round, smooth, and cream-colored.

Results:

● After 12-14 hours of incubation in LB broth, turbidity indicates successful bacterial growth. The
bacteria should be in the log phase of growth, which is ideal for experiments like plasmid
extraction.
● If the bacteria were streaked on LB agar plates, individual colonies should be visible after 16-24
hours of incubation at 37°C. These colonies are typically small, round, and creamy-white in
appearance, and they may exhibit smooth edges.

[4]
Discussion:

● LB Medium Composition: The LB medium is a rich, non-selective medium that supports the
growth of a wide range of bacteria, including Escherichia coli. The tryptone provides peptides
and amino acids that are essential for protein synthesis, while yeast extract supplies vitamins and
minerals. The NaCl helps maintain osmotic balance in the medium.
● Growth of E. coli in LB Media: E. coli grows rapidly in LB broth, as it is a rich medium that
meets most of the bacterium's nutritional needs. The bacterium will enter the log phase of growth
within 4-6 hours in LB broth under optimal conditions (37°C with shaking). The turbidity of the
culture increases as the bacterial population grows. This makes LB broth an excellent medium.
● Experimental Use: LB broth is commonly used for cloning, gene expression, and plasmid
propagation in laboratory settings.

Precautions:

1. Sterility:
○ Always use sterile techniques to avoid contamination. Ensure all equipment (pipettes,
loops, etc.) are sterile before use.
2. Handling Bacteria:
○ Handle bacteria with care and always wear gloves while handling .
3. Autoclaving:
○ Ensure proper autoclaving to sterilize the LB media. Failure to properly sterilize may lead
to contamination.
4. pH Monitoring:
○ Ensure that the pH of the medium is adjusted to 7.0 before autoclaving, as an incorrect
pH can hinder bacterial growth.
5. Incubation:
○ Ensure the incubation temperature is strictly maintained at 37°C. Temperatures higher
or lower may lead to poor bacterial growth or stress the cells.
6. Storage:

○ If you plan to store the LB broth or agar plates, make sure they are stored properly to
prevent contamination. Liquid LB broth can be stored at 4°C for short periods (1-2
weeks), while agar plates should be stored upside down with labeling name of species
,date of making to prevent condensation from interfering with bacterial growth.

[5]
Experiment 1: Isolation of Plasmid DNA from E. coli and
Quantification Using Agarose Gel Electrophoresis.

INTRODUCTION

In this experiment, the isolation of plasmid and genomic DNA from Escherichia coli (E. coli) is
implicated. E. coli is broadly used in molecular biology since it can easily be cultured and handled
besides, it can carry plasmids; small, circular DNA molecules existing apart from the chromosomal DNA.
Plasmids find their wide application in genetic engineering and cloning experiments.

We then measure the isolated DNA using agarose gel electrophoresis, a technique that separates nucleic
acids on the bases of size and charge. Agarose gel electrophoresis is very useful for the verification of
DNA purity and quantity and the determination of the size of DNA fragments. We observe DNA bands
under UV light after staining with a dye that binds to nucleic acids.

MATERIALS

● E. coli culture (overnight grown)

● Lysis buffer (for plasmid extraction) SDS buffer
● Resuspension buffer - Glucose Tris EDTA buffer
● isopropanol (for DNA purification)
● Agarose gel electrophoresis apparatus
● DNA ladder (size marker)
● Ethidium bromide
● Micropipettes and tips
● 1X TAE or TBE buffer
● Bromophenol Blue
● UV transilluminator
● Sterile distilled water
● Centrifuge and microcentrifuge tubes
● 1 Litre of 10X-TAE Buffer [(48.5 g tris base in 900 ml double-distilled H2O; 11.4 ml
glacial acetic acid; 20 ml 0.5 M EDTA solution (pH 8.0)]. Adjust volume to 1 L
● 1 Litre of 10X TBE Buffer [108 g tris base; 55 g boric acid; 900 ml double-distilled
H2O; 40 ml 0.5 M EDTA solution (pH 8.0)- Adjust volume to 1 L
● 6X gel-loading dye: Bromophenol blue 0.25% (w/v), Xylene cyanol 0.25% (w/v),
Sucrose in water 40% (w/v)

[6]
Principle:
Isolation of genomic DNA is based on stepwise subtractive elimination of all macromolecules except
DNA. EDTA and SDS is used to lyse the bacterial cell wall and membrane. Glucose is used to maintain
the osmolarity of DNA. Tris-Cl helps in maintaining slightly alkaline pH to prevent DNA from being
degraded. EDTA removes Mg which is essential for structural stability of cell wall. It also inhibits activity
of DNAases. SDS solubilises cell membrane and denatures proteins. DNA is precipitated in cold ethanol
or isopropanol. Sodium ions from NaCl bind to phosphate groups and thus helps in precipitating DNA.

Plasmids are small supercoiled covalently closed circular DNA molecules and can be separated from
the larger bacterial chromosomes. EDTA acts as a chelating agent and chelates divalent cations.
Divalent cations like are required for maintenance of integrity of bacterial cell wall and helps in activity
of DNase. Thus chelation of divalent ions by EDTA prevents activity of DNases. Glucose is used to
maintain the osmolarity of DNA.

Soution 2 contains NaOH and SDS. Sodium dodecyl sulphate is a detergent which causes lysis of the
cell membrane and it denatures protein. NAOH provides alkaline conditions which disrupts the
hydrogen bonds between DNA bases thus converting the dsDNA to ssDNA.This process of
denaturation in presence of NAOH is called alkaline lysis. Potassium acetate decreases the alkalinity
thereby ensuring reformation of hydrogen bonds between the ssDNA to dsDNA. Since plasmid DNA is
covalently closed DNA it renatures to correct conformation however the genomic DNA forms
precipates due to random annealing and thus can be separated by centrifugation.

[7]
PROCEDURE
Genomic DNA isolation

1. Took 1 mL of E. coli growing in LB medium in a microcentrifuge tube.

2. Centrifuged at 5000 rpm for 5 minutes to obtain a pellet.

3. Decanted the supernatant.

4. Added 200 µL of GTE (Glucose, Tris-Cl, EDTA) buffer. Resuspended the pellet by breaking or
vortexing it. Alternatively, resuspended the pellet by pipetting.

5. Incubated at room temperature for 5 minutes.

6. Added 400 µL of 1% SDS, mixed by inversion, and kept at room temperature for 5 minutes.

7. Added 120 µL of 5M NaCl. Mixed gently by inversion.

8. Centrifuged at 10,000 rpm for 15 minutes.

9. Carefully pipetted out the supernatant without disturbing the pellet and transferred it to a fresh
microfuge tube.

10. Added an equal volume of chilled isopropanol and mixed by gentle inversion.

11. Centrifuged at 10,000 rpm for 15 minutes.

12. Decanted the supernatant and air-dried the pellet.

13. Dissolved the DNA pellet in 30-50 µL of sterile double-distilled water.

[8]
A. Plasmid DNA Isolation:

1. Harvest Cells: Collect E. coli cells (from overnight culture) by centrifugation at 10,000 rpm for
10 minutes from 2 ml media .
2. Resuspend Cells: Resuspend the pellet in a small amount of resuspension buffer which is 100
microlitre Glucose +Tris +EDTA GTA (Glucose maintain osmoregularity ) and wait for 5 min in
room temperature
3. Cell Lysis: Add 200 microlitre lysis buffer and gently mix to lyse the cells. Incubate for 5
minutes at room temperature. The Lysis buffer we use is SDS.
4. Neutralization: Add a 150 microlitre neutralization buffer which is 3 Molar potassium acetate .
to stop the lysis process. Centrifuge the mixture 12000 rpm for 12 minutes to separate the plasmid
DNA from the cellular debris.
5. Plasmid Purification: Transfer the supernatant (containing plasmid DNA) to a new tube and mix
with equal volume of isopropanol and finally centrifuge at 10K rpm for 5 min .Discard the
supernatant and wash the pellet .

[9]
B. DNA Quantification by Agarose Gel Electrophoresis:

1. Prepare an agarose gel (0.8-1%) by dissolving agarose in 1X TAE buffer and adding a DNA stain
(e.g., ethidium bromide)
2. Heat the mixture until it looks clean and cool about 50 to 60%.
3. Load the DNA samples (plasmid DNA with LOading Dye - bromophenol Blue) into the gel
wells, along with a DNA ladder (size marker) for comparison.
4. Run the electrophoresis at 100-120 V for about 30 minutes, or until the dye has moved
sufficiently through the gel.
5. Visualize the gel under UV light or using a gel imaging system (transilluminator ) to assess the
size and quantity of the DNA.

OBSERVATION AND RESULT

Isolation of Genomic DNA

 Genomic DNA is viscous and clear liquid.

 On agarose gel, genomic DNA migrates as a high molecular weight band near the top.

 A faint or smeared band may indicate degradation or poor extraction.

Plasmid DNA Isolation

 Plasmid DNA is clear and slightly viscous.

 On the gel, two bands are normally observed with plasmid DNA: a fast-migrating supercoiled
form (preferred) and a slower-migrating relaxed form.

 A well-defined, prominent supercoiled band denotes the successful isolation.

Agarose Gel Electrophoresis

 DNA Genomic migrates slowly since it is a big DNA molecule while plasmid shows multiple
forms such as supercoiled and relaxed.

 Band intensity corresponds to the amount of DNA; sharp strong bands indicate higher yields.

[10]
Quantifying DNA

 The intensity of the band on the gel can be compared using DNA ladder for an approximation of a
quantity

Contamination Check

 Presence of additional bands or smear of the DNA indicates contamination, such as having RNA.

 Good DNA preparations should have evident, clear bands without extra smears.

CONCLUSION

Plasmid DNA from E. coli is confirmed through gel electrophoresis. The plasmid DNA should show a
distinct band, while the genomic DNA should appear as a broader smear, providing a clear differentiation
between the two types of DNA.

Precautions
1. Cultures must be raised in aseptic conditions.

2. Microfuge tubes, tips etc should be autoclaved before use.

3. Resuspending of pellets in GTE must be thorough.

4. The supernatant must be carefully pipetted out without disturbing the pellet.

5. Before adding ddwater ensure the pellet should be completely dry with no residual isopropanol.

[11]
Experiment 2: Isolation of Genomic DNA from Plant
Samples Using CTAB Method

Introduction: The isolation of high-quality genomic DNA from plant tissues is a critical step in molecular
biology studies, including genetic analysis, PCR, and cloning. Plant cell walls, which are rigid and
complex, make DNA extraction more challenging compared to animal cells. The CTAB (Cetyltri methyl
ammonium Bromide) method is widely used for extracting genomic DNA from plant tissues. This
method uses a detergent (CTAB) to break down the cell membrane and a salt solution to remove
impurities like proteins and lipids. The resulting DNA is then purified and can be used for downstream
applications.

Materials Required:

● Plant tissue (e.g., Spinach leaves, cauliflower)

● Pestle and mortar
● CTAB extraction buffer {2% CTAB +1.4M Nacl +20mM Nacl+100mM tris HCL}
● Isoamyl alcohol (24:1)
● Isopropanol
● 70% ethanol
● Centrifuge and microcentrifuge tubes
● Water bath
● DNA ladder (for later gel analysis)
● Agarose gel electrophoresis equipment

Principle:

The CTAB method for plant genomic DNA extraction works by breaking down the plant cell wall and
membrane to release the DNA. The CTAB reagent, a detergent, helps to lyse the cells and solubilize the
lipids and proteins in the plant tissue. The addition of a high-salt buffer helps to precipitate out
polysaccharides, which are abundant in plant cells and can interfere with DNA purification. The DNA is
then purified by isoamyl alcohol, which removes proteins and other contaminants. The final DNA is
precipitated with isopropanol and can be dissolved in a buffer for further use

[12]
Procedure:

1. Sample Preparation: Approximately 100 micrograms of fresh plant tissue (e.g., spinach leaves
and cauliflower) were taken.

2. Tissue Grinding: The tissue was ground into a fine powder using a mortar and pestle or a
homogenizer in the presence of liquid nitrogen.

3. Lysis: The powdered tissue was transferred to a tube containing a pre-warmed 200 microliter
CTAB buffer. It was then incubated at 65°C for 30 minutes to allow the detergent to break down
the cell walls and membranes.

4. Phase Separation: After incubation, an equal volume of chloroform or isoamyl alcohol (24:1)
was added. The mixture was gently mixed by inversion and then centrifuged at 10,000 rpm for 10
minutes. This step separated the aqueous phase (containing DNA) from the organic phase
(containing proteins and lipids).

5. DNA Precipitation: The upper aqueous phase was transferred to a new tube with the help of a 1
ml micropipette, and 0.6 volumes of chilled isopropanol were added. The mixture was gently
mixed to precipitate the DNA. The solution was incubated at -20°C for 30 minutes to enhance
precipitation.

6. DNA Washing: The mixture was centrifuged at 12,000 rpm for 10 minutes to collect the DNA
pellet. The pellet was washed with 70% ethanol to remove remaining contaminants and then air-
dried.

7. DNA Rehydration: The DNA pellet was dissolved in an appropriate volume of TE buffer or
distilled water for further use.

[13]
Observations:

● After the CTAB treatment, the solution should appear viscous, indicating the presence of DNA.
● The separated aqueous phase should be clear after centrifugation, and the DNA should precipitate
as a white pellet upon adding isopropanol.
● The DNA should be visible as a band after electrophoresis if successful.

Results:

● The quantity and quality of the isolated DNA can be assessed by running an aliquot on an agarose
gel alongside a DNA ladder. The gel should show a single, clear band for high-quality genomic
DNA.
● The intensity of the band can be used to estimate the concentration of the DNA.

Discussion:

● The CTAB method efficiently isolates genomic DNA from plant tissues, even from those with
complex secondary metabolites or large amounts of polysaccharides.
● If the DNA appears degraded or there are multiple bands on the gel, it could indicate issues with
the lysis procedure or contamination with other molecules, such as RNA or phenolic compounds.
● The use of CTAB ensures effective removal of polysaccharides, which are common contaminants
in plant DNA extraction.

Conclusion:

● The CTAB method is a reliable and widely used technique for isolating high-quality genomic
DNA from plant tissues. The DNA extracted can be used in various applications, such as PCR,
genetic analysis, and cloning.

[14]
Experiment 3: Quantification of Unknown DNA by
Diphenylamine Reagent (Colorimetry).

Introduction:

DNA quantification is one of the most significant steps in molecular biology, as accurate amounts of
DNA are required for correct utilization in downstream applications like PCR, cloning, or sequencing.
One of the most enduring yet reliable methods to quantify DNA is the colorimetric Diphenylamine (DPA)
technique. The method is based on the reaction between the sugar moiety, deoxyribose, which is a part of
DNA with diphenylamine and goes on to change the colour. The intensity of the color directly varies with
the amount of DNA and can be quantitatively measured using a spectrophotometer.

Principle:

The Diphenylamine reagent reacts with the deoxyribose in DNA to form a blue-colored complex. The
intensity of the blue color is directly proportional to the concentration of DNA in the sample. By
comparing the absorbance of the sample to a standard curve made with known concentrations of DNA,
the concentration of the unknown DNA sample can be determined.

Materials:

● Herring sperm DNA samples (or samples with varying DNA concentrations for standard curve)
● Diphenylamine reagent
● Standard DNA (e.g., calf thymus DNA or a known DNA concentration)
● Test tubes or cuvettes
● Spectrophotometer (or colorimeter)
● Distilled water
● Pipettes
● Vortex mixer (optional)

Reagents:

● Diphenylamine reagent:
○ 2% diphenylamine in glacial acetic acid (prepare fresh if necessary)
○ Acetic acid (solvent)
○ Sulfuric acid (concentrated)
● Note: Prepare the diphenylamine reagent by dissolving 2g of diphenylamine in 100 mL of glacial
acetic acid, and adding a small amount of concentrated sulfuric acid.

[15]
Procedure:

1. Preparation of Standards and Samples:

○ Prepare a series of standard DNA solutions of known concentrations by diluting the
standard DNA stock in distilled water. A typical concentration range for standards might
be from 0,100,200,400,600 and 800 µg/2mL.
○ Label the test tubes or cuvettes for the standard solutions and the unknown sample(s) A
and B.
2. Reaction Setup:
○ To each test tube, add 2 mL of the standard DNA solution (or the unknown DNA sample)
and 4 mL of diphenylamine reagent.
○ Mix the contents gently by vortexing or swirling the test tube.
○ Allow the reaction to proceed at room temperature for 30 minutes to 1 hour, or until the
color develops fully. The reaction will produce a blue color, the intensity of which will
increase with the amount of DNA present.
3. Blank:
○ Prepare a blank by adding 2 mL of distilled water (instead of DNA solution) to 4 mL of
diphenylamine reagent. This will account for any background color due to the reagent.
4. Color Measurement:
○ After the reaction has completed and the color has developed, measure the absorbance of
each sample (including the blank) at 600 nm using a spectrophotometer or colorimeter.
The absorbance at this wavelength corresponds to the intensity of the blue color, which is
directly proportional to the DNA concentration.
5. Plotting the Standard Curve:
○ Plot the absorbance values of the standard DNA solutions against their known
concentrations to create a standard curve.
○ Ensure the standard curve is linear (typically between 0-100 µg/mL DNA). If the curve is
nonlinear, dilute the unknown sample accordingly.

[16]
6. Determination of DNA Concentration in Unknown Samples:

○ Using the absorbance value obtained for the unknown DNA sample, interpolate its
concentration from the standard curve.

[17]
Data Analysis:

● Calculate the concentration of DNA in the unknown sample by comparing its absorbance to the
standard curve.

● If the absorbance falls outside the range of the standard curve, dilute the sample and repeat the
measurements.

● Safety Considerations:

● Wear gloves, lab coat, and safety glasses to avoid contact with chemicals (especially sulfuric acid
and diphenylamine reagent).
● Handle concentrated sulfuric acid with care, as it is highly corrosive.
● Dispose of waste according to laboratory guidelines for hazardous chemicals.

[18]
Precautions:

● 1.Glassware should be clean and dry.

● 2.Test tubes should be properly labeled.
● 3.DNA stock solution and DPA reagent should be freshly prepared.
● 4.DNA dilutions should be prepared accurately.
● 5.Mix the solutions thoroughly before keeping them in a water bath.
● 6.Incubation time for all the test tubes should be the same.
● 7.DPA should be handled carefully because it contains acid.
● 8.Set zero of the colorimeter using blank before measuring absorbance.
● 9.Cuvette must be wiped with tissue paper to remove fingerprints and any solution sticking
outside.

Conclusion:

This experiment allows for the quantification of DNA in an unknown sample by measuring the color
change resulting from the diphenylamine reaction. By using a standard curve, the DNA concentration of
unknown samples can be accurately determined.

[19]
Experiment 5 : Control of Translation in Escherichia coli Using
Prokaryotic Inhibitors (Kanamycin / Streptomycin).

Objective:

To study the effect of translation inhibitors (Kanamycin or Streptomycin) on the growth of Escherichia
coli by measuring the optical density (OD) at various time intervals and plotting the growth curve. This
experiment will help understand how these antibiotics affect protein synthesis and bacterial growth.

Materials:

● LB Media (Luria-Bertani Broth)

● Escherichia coli strain (e.g., DH5α or any standard lab strain)
● Kanamycin (antibiotic)
● Sterile test tubes (3 × 20 mL)
● Spectrophotometer (for measuring OD at 600 nm)
● Incubator (37°C)
● Pipettes and sterile tips

Experimental Setup and Procedure:

1 ) Preparation of Test Tubes:

● Label three test tubes:

● Test Tube 1 (Blank): 20 mL of LB media with E. coli inoculated.
● Test Tube 2 (LB + Kanamycin): 20 mL of LB media with E. coli and Kanamycin added.
● Test Tube 3 (LB + Kanamycin at 60 minutes): 20 mL of LB media with E. coli, but
Kanamycin will be added only after 60 minutes.

[20]
(2) -Inoculation of Bacteria:

● Add 1–2 mL of fresh E. coli culture to each of the test tubes (Test Tubes 1, 2, and 3) under sterile
conditions.
● The final bacterial concentration will be low, just enough to start growth, typically around 10^6
CFU/mL.

(3)-Incubation:

● Place all three test tubes in a 37°C incubator and allow the cultures to grow for 30 minutes.
● During the first 30 minutes, Test Tubes 1 and 3 bacteria grow in the presence or absence of
Kanamycin. Tube no 2 growth is restricted because of kanamycin .Test Tube 3 will remain
without Kanamycin until the 60-minute mark.

(4)-Addition of Kanamycin:

● After 60 minutes, add Kanamycin (final concentration: 30–50 µg/mL) Test Tube 3.
● Test Tube 1 remains as the control with no antibiotic.

(5)-OD Measurement:

After adding Kanamycin to Test Tubes 3, take OD readings at 600 nm (OD600) at the following time
points:

● Time 0 (T0): Before the addition of Kanamycin (after 60 minutes of incubation).

● Time 1 (T1): 30 minutes after adding Kanamycin.
● Time 2 (T2): 60 minutes after adding Kanamycin.
● Time 3 (T3): 90 minutes after adding Kanamycin.
● Time 4 (T4): 120 minutes after adding Kanamycin.
● Time 5 (T5) : 150 minutes after adding Kanamycin

Time OD600 (Test Tube 1: OD600 (Test Tube 2: OD600 (Test Tube 3: Kanamycin
(minutes) Blank, Control) Kanamycin from start) added after 60 minutes)
0 0.05 0.05 0.05
30 0.1 0.08 0.1
60 0.2 0.1 0.15
90 0.35 0.12 0.1
120 0.5 0.14 0.05

[21]
6 -Plotting the Growth Curve:

After measuring the OD600 at the specified time points, plot a growth curve:

● The x-axis represents time (in minutes).

● The y-axis represents the OD600 readings, which indicates bacterial growth (higher OD means
more bacteria).

X = TIME

Y= OD VALUE
[22]
Observations:

1)- Test Tube 1 (Blank – LB + E. coli):

● As the bacteria grow, the OD600 will increase steadily over time as the bacteria multiply in the
LB media.
● This represents normal growth with no antibiotic interference.

2)- Test Tube 2 (LB + Kanamycin):

● After Kanamycin is added, the OD600 will increase initially but should start to plateau or show
slower growth after some time.
● Kanamycin inhibits bacterial protein synthesis, so bacterial growth will be significantly reduced,
especially as the drug starts taking effect.

3)-Test Tube 3 (LB + E. coli and Kanamycin added after 60 minutes):

● Initially, the bacteria will grow normally for the first 60 minutes (without Kanamycin), so the
OD600 will increase.
● After the addition of Kanamycin at the 60-minute mark, the growth will slow down or stop,
showing a significant decrease in the rate of bacterial growth.

Results:

● Test Tube 1 (Blank – No Antibiotic):

○ As expected, this test tube shows normal growth, with a steady increase in OD600,
indicating active bacterial replication and protein synthesis.
● Test Tube 2 (Kanamycin from the beginning):
○ After Kanamycin is added, bacterial growth should slow down or stop, showing a
flattening or decrease in OD600 after some time. This demonstrates that Kanamycin is
inhibiting protein synthesis and halting bacterial proliferation.
● Test Tube 3 (Kanamycin after 60 minutes):
○ Initially, you will see normal growth in Test Tube 3, similar to the control (Test Tube 1),
but after the addition of Kanamycin at 60 minutes, the growth rate will significantly slow
or plateau.

[23]
Discussion:

❖ Effect of Kanamycin: Kanamycin works by binding to the 30S ribosomal subunit of the
bacterial ribosome, preventing protein synthesis. The results from this experiment should show a
clear reduction in bacterial growth on Kanamycin treatment.
❖ Test Tube 1 (control) will show typical bacterial growth, as no antibiotic is present to interfere
with protein synthesis.
❖ Test Tube 2 (Kanamycin from the start) will show a slower growth curve compared to the
control, demonstrating how Kanamycin inhibits bacterial translation.
❖ Test Tube 3 (Kanamycin added at 60 minutes) will show normal growth for the first 60 minutes,
after which the antibiotic will begin to inhibit protein synthesis and bacterial growth.
❖ Effect of Time: The timing of Kanamycin's addition is crucial. In Test Tube 3, where
Kanamycin is added after 60 minutes, bacteria grow initially because translation is not inhibited.
However, once Kanamycin is introduced, the growth slows or stops as translation is inhibited,
and the bacteria can no longer produce proteins essential for growth.

[24]
Precautions:

1. Sterile Technique:
○ Ensure that all equipment (pipettes, test tubes, etc.) is sterile to prevent contamination,
which could affect the experiment's results.
2. Accurate OD Measurements:
○ Ensure proper calibration of the spectrophotometer before measuring OD600. Take
measurements at the same wavelength (600 nm) each time.
3. Kanamycin Concentration:
○ Use the appropriate Kanamycin concentration (30-50 µg/mL) for your specific E. coli
strain. Higher concentrations could completely inhibit bacterial growth, while lower
concentrations may not show a significant effect.
4. Incubation Conditions:
○ Ensure the incubator is set to 37°C, the optimal temperature for E. coli growth.
5. Timing of Measurements:
○ Ensure you are taking OD readings at the correct intervals (every 30 minutes) and at
consistent times to generate an accurate growth curve.

CONCLUSION
This experiment allows you to visually and quantitatively assess how translation inhibitors like
Kanamycin affect bacterial growth. By plotting the growth curve and comparing the effects of
Kanamycin, you can draw conclusions about the antibiotic's role in inhibiting protein synthesis and its
overall impact on bacterial proliferation.

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