JYOTI NIVAS COLLEGE AUTONOMOUS

WORKSHOP REPORT
AT
AZYME BIOSCIENCES PVT. LTD.
Submitted to the Department of Genetics in partial fulfilment of the requirement for the award
of the Bachelor of Science programme.

Submitted by

Ms. Srijani Ghosh

Reg. No. 22BTGT016H

JYOTI NIVAS COLLEGE AUTONOMOUS

BENGALURU – 560095

July 2024
 CERTIFICATE

This is to certify that Ms. Srijani Ghosh has successfully completed the workshop Training in
Molecular Techniques at Azyme Biosciences Pvt. Ltd for the Final year
B. Sc BTGT 5th semester course for the year 2024-2025 in partial fulfilment of the
requirements for the award of Bachelor of Science programme.

Teacher incharge Head of the Department

Internal Examiner

External Examiner
 TABLE OF CONTENTS:

Topic Page No.

Introduction 4

SDS PAGE 5
Western Blotting 10

Study of gene regulation in E coli by visual screening 12

method
Isolation of RNA from leaf sample 14

PCR 16
Agarose Gel Electrophoresis 18

Isolation of genomic DNA from prokaryotic source 20

 INTRODUCTION

PAPER V - GENE REGULATION AND DNA REPAIR

AZYME BIOSCIENCES
Dedicated to serving the scientific community through contract research, Azyme Biosciences Pvt. Ltd. is an ISO
9001-2008 Certified Research Laboratory that was founded in 2008 with the goal of bridging the gap between
academia and industry. The company was founded as a result of using an entrepreneurial attitude and directing
resources and energy toward prospect creation. Training in a variety of fields, including HPLC, Food
Biotechnology, Biotechnology Instrumentation, Biotechnology Projects and Training, Animal Cell Culture
Training, and Biotechnology Corporate Training Programs in Bangalore, is one of Azyme's key initiatives in
enhancing students' and researchers' critical thinking skills and preparing them for the workforce.

They give students the chance to work in a state-of-the-art research setting before they pursue careers.Azyme
Biosciences has been set up with the prime idea of skill development. It is an organisation that truly connects
academia and research with industrial practices. Azyme Biosciences is a consortium of experienced scientists and
research scholars having state-of-the-art laboratory infrastructure with the use of the newest and most innovative
research methodologies. It offers students an opportunity to experience a cutting-edge research environment
before pursuing their careers and providing scientific laboratory services.

LEARNING OUTCOMES

● Preparation of protocol
● Result Interpretation
● Troubleshooting techniques
● Individual handling of techniques and equipment
● An understanding of sample preparation methods

OVERVIEW OF THE EXPERIMENTS PERFORMED

Understanding of gene expression or regulation was made possible through Molecular Biology

Such experiments are gene regulation studies in Escherichia coli (E. coli), RNA isolation from leaf samples,
Polymerase Chain Reaction (PCR) and Agarose Gel Electrophoresis (AGE) and Genomic DNA extraction.
Gene regulation in E. coli constitutes the fundamental basis for prokaryotic gene expression understanding.
Isolation of RNA from leaf samples is an important step in gene expression studies. This procedure leads to the
purification of intact, high-quality RNA suitable for reverse transcription and quantitative PCR. The quality of
the RNA isolates is very important for reproducible and reliable data in gene expression analysis . Polymerase
Chain Reaction (PCR) is frequently used to amplify discrete DNA sequences. This method is used for lots of
things such as cloning, sequencing and diagnostic testing. Through AGE, it is possible to see and evaluate the
results of a DNA sample, which can be very helpful in order to obtain information about its quality as well as
quantity regarding amplified products. AGE is an important checking step for successful result of PCR other
DNA manipulations.
EXPERIMENTS:

SDS-PAGE

AIM:

To separate and detect the proteins present in the given sample using SDS-PAGE and western blotting.

PRINCIPLE:

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a commonly used technique in

laboratories to separate proteins by their molecular weight. In this method, proteins are first treated with Sodium
Dodecyl Sulphate (SDS), a denaturing detergent and provides a uniform negative charge. This treatment ensures
all proteins have a similar charge-to-mass ratio. When an electric field is applied, the negatively charged
proteins migrate towards the positively charged electrode, with smaller proteins moving faster through the gel
than larger ones, thus achieving separation based on size. It is a technique widely used in forensics, genetics,
biotechnology and molecular biology to separate the protein

MATERIALS REQUIRED:

REAGENTS:

1) Electrode buffer (pH 8.2)

● Tris HCl - 6g
● Glycine - 14.4g
● SDS - 1g
● Distilled water - 1000ml

2) Separating gel buffer (pH 8.8)

● Tris HCl (1.5M) – 2.36g in 10mL of distilled water

3) Stacking gel buffer (pH 6.8)

● Tris HCl – 1.57g in 10mL of distilled water

4) Potassium persulphate

● Dissolve 0.1g of potassium persulphate in 1 ml of distilled water

5) Sodium dodecyl sulphate (SDS)

● Dissolve 0.1g of SDS in 1ml of distilled water

6) Stock solution

● 30% Acrylamide and 0.8% bis acrylamide dissolved in 100 ml of distilled water.

7) SDS Loading Dye

● Bromophenol Blue – 0.025%

● SDS – 10%
● β - Mercaptoethanol – 0.5ml
● Glycerol – 30%
● Tris HCl (0.1M) (pH 8.2) – 0.5ml

8) Staining Solution

● Methanol – 40ml
● Distilled water – 55ml
● Acetic acid – 5ml
● Coomassie Brilliant Blue – 0.01g

9) De-staining Solution

● Methanol – 40ml
● Distilled water – 55ml
● Acetic acid – 5ml

OTHER REQUIREMENTS:

Electrophoresis chamber, water bath, micropipettes and tips, comb, gel casting tray, nitrocellulose membrane,
filter paper, sponge.

PROCEDURE:

DAY 1:

1. Gel Preparation:

- Prepare the acrylamide solution for the separating gel.

- Add polymerization catalysts (e.g., TEMED and ammonium persulfate) and pour the gel mixture into the
casting frame.

- Overlay with isopropanol or water to avoid air bubbles. Allow it to polymerize.

2. Stacking Gel Preparation:

- After the separating gel polymerizes, pour off the overlay liquid.
 - Prepare the acrylamide solution for the stacking gel.

- Insert the comb into the gel solution to create wells and allow it to polymerize.

3. Buffer Preparation:

- Prepare the running buffer (typically Tris-Glycine-SDS buffer) for the electrophoresis tank.

4. Sample Preparation:

- Mix protein samples with SDS sample buffer containing SDS, reducing agents, glycerol, and tracking dye.

- Heat the mixture at 95°C for 5 minutes to denature the proteins.

5. Loading Samples:

- Carefully remove the comb from the polymerized stacking gel.

- Rinse the wells with running buffer to remove any unpolymerized acrylamide.

- Load the prepared protein samples and molecular weight markers into the wells using a micropipette.

6. Electrophoresis Setup:

- Assemble the gel into the electrophoresis tank and fill it with running buffer.

- Ensure the gel is properly positioned, and the buffer covers the gel wells.

7. Separation of gel :

-The separating gel mixture and the stacking gel mixture are prepared and subjected to ultrasonic treatment
for 5 minutes.

8. Separating gel mixture:

-To the separating gel mixture, 100μL of SDS, 50μL of ammonium persulfate, and 10μL of TEMED are
added. Upon adding TEMED, the gel mixture is immediately mixed and poured into the setup until it reaches
75% of the total length. It is then left undisturbed for a few minutes to solidify.

9. Stacking gel mixture :

-To the stacking gel mixture, 100μL of SDS, 50μL of ammonium persulfate, and 10μL of TEMED are
added. As soon as TEMED is introduced, the gel mixture is thoroughly mixed and poured into the setup until it
reaches the top. A comb is then carefully inserted, making sure it does not touch the separating gel. The setup is
left undisturbed for a few minutes to allow solidification.

10. Solidification:

- Once the gel has solidified, the comb is gently removed, and the wells are marked for easy identification.

11. The bottom spacer of the gel slab is taken off, and the setup is placed into the electrophoretic unit with the
electrode buffer. The marker protein is loaded into the first well. In an Eppendorf tube, 50μL of the sample is
mixed with 50μL of SDS gel loading dye and denatured in a boiling water bath for 10 minutes. Then, 50μL of
the sample is loaded into the designated wells.

12. The electrical connections are established, the voltage is set to 50, and the instrument is turned on.
Electrophoresis is run for approximately 4 hours, until the bands have migrated 75-80% of the gel slab's length.

Visualization of the Bands:

1. Once electrophoresis was done, the gel unit was obtained

2. The gel is placed in 100mL of staining solution overnight.
3. The next day, a destaining solution was added and was repeated 2-3 times, until the Bands were
clear.

OBSERVATION:

On staining SDS-Polyacrylamide gel, different proteins appeared as dark blue bands against a light background.

RESULT:

The different proteins present in the sample were separated using SDS-PAGE.

DISCUSSION:

SDS-PAGE is a valuable technique for analyzing protein mixtures by separating them according to size. This
approach is highly effective for assessing protein structure, function, and purity. However, some challenges may
arise during the experiment, such as uneven sample loading and incomplete gel polymerization, which can
impact the resolution of the protein bands. To mitigate these issues, it is crucial to ensure thorough
polymerization, carefully load samples, and prepare buffers correctly. Protein characterization, purity analysis,
and molecular weight determination are essential in both research and diagnostic settings.
CONCLUSION

SDS-PAGE is a reliable technique for protein separation, offering insights into protein composition and
molecular weight. Despite certain limitations like incomplete polymerization and uneven sample loading,
adhering to proper protocols and optimization strategies can yield clear and accurate results. Its applications in
research and diagnostics underscore its importance as a fundamental tool in molecular biology.
 WESTERN BLOTTING
AIM:

To detect and evaluate specific proteins in a sample using Western blotting.

PRINCIPLE:

Western blotting is a technique that combines protein electrophoresis and antibody binding to detect target
proteins with high sensitivity. It exploits the inherent specificity of antigen-antibody interaction to identify
specific antigens using polyclonal or monoclonal antibodies. The technique requires the use of several
components which include an SDS-PAGE gel to separate proteins based on size and a nitrocellulose membrane
to immobilise the proteins after electrophoresis. A primary antibody specific to the target protein is then used,
followed by a secondary antibody conjugated with an enzyme that binds to the first antibody. To avoid non-
specific binding, a blocking buffer is used. Finally, a substrate is added, which combines with the enzyme to
create a signal that can be measured (chemiluminescent or colorimetric). These components work together to
provide a clear and specific identification of the target protein.

MATERIALS REQUIRED:

REAGENTS

Washing Solution - Phosphate Buffer Saline (PBS) (pH 7)

1. Disodium hydrogen orthophosphate - 1.44g

2. Potassium dihydrogen orthophosphate - 0.24g
3. Sodium chloride - 8g
4. Potassium Chloride - 0.2g
5. Distilled water - 1000ml

PROCEDURE

DAY - 1

1. After electrophoresis, the nitrocellulose membrane was taken out and placed in a petri plate.
2. 10 ml of the PBS buffer and 0.3g of Cassini were added and kept overnight.

DAY - 2

3. The solution was discarded and the membrane was washed with 10 ml of PBS and 10μl of Primary
antibody was added to the membrane for 10 minutes.
4. The solution was discarded and the membrane was washed with 10 ml of PBS and 100μl of Triton X.
5. The solution was discarded and washed with 10 ml of PBS.
 6. The solution was discarded, and 10 ml of PBS and 3μl of the Secondary Antibody was added to the
membrane for 10 minutes.
7. The solution was discarded and washed with 10 ml of PBS and 100 ml Triton X.
8. The solution was discarded, and washed with 10 ml of PBS.
9. The membrane was then kept in a solution that contained 10 ml PBS, 10 ml Guaiacol, and 10 μl Hydrogen
peroxide.

OBSERVATION:

Yellow-coloured bands were observed.

RESULT:

When an enzyme-conjugated secondary antibody was used for western blotting, yellow bands appeared on the
membrane where the target proteins were situated. The intensity of the bands indicates the amount of protein
present which could be detected using a spectrophotometer.

DISCUSSION

Western blotting is a complex protein analysis technique that combines electrophoresis with antibody binding.
Common issues include inadequate protein transfer, weak signals, non-specific binding, and strong background
noise. Preventive measures include ensuring ideal transfer circumstances, utilizing high-quality antibodies, and
blocking properly. Western blotting has numerous applications, including medical diagnostics, protein function
research, and disease marker detection. Despite these obstacles, the approach provides great sensitivity and
specificity.

CONCLUSION

Western blotting remains a critical technique for detecting individual proteins, with its high sensitivity and
specificity contributing to significant advancements in research and diagnostics. By overcoming issues like non-
specific binding and transfer efficiency, the results can be greatly improved, reinforcing the technique's
indispensability in protein analysis and biomarker discovery.
 STUDY OF GENE REGULATION IN E.coli CELLS BY VISUAL SCREENING
METHOD
AIM:

The main objective of the experiment is to study the gene regulation in E.coli cells by the blue-white visual
screening method.

PRINCIPLE:

Gene regulation in E. coli cells is majorly controlled by the Lac operon. It has 3 structural genes namely lac z,
lac y, and lac a. Lac z encodes for β-galactosidase enzyme which catalyzes the hydrolysis of lactose into
glucose and galactose. Visual screening of the cells is made possible due to the use of chemicals like IPTG
(Isopropyl β-d-1-thiogalactopyranoside) and X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside). IPTG
acts as the initiator of the lac operon by removing the repressor from the lac operon and inducing gene
expression. X-gal is a chromogenic substance which in the presence of β-galactosidase gets hydrolyzed to
produce the blue colour to the colonies.

MATERIALS REQUIRED:

EQUIPMENTS:

Micropipette, Sterilised Micropipette tips, Sterilised Eppendorf tubes, centrifuge, Laminar Air Flow chamber,
refrigerator.

REAGENTS:

1. M9 Broth
● Disodium hydrogen orthophosphate Na 2 P04 - 0.15g
● Sodium chloride NaCl- 0.12g
● Potassium dihydrogen orthophosphate K2H2 PO4 - 0.74g
● Ammonium chloride NH 4Cl - 0.10g
● Peptone - a pinch
● Magnesium chloride MgCl2 - 0.002g
● Distilled water - 25ml
2. X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)
3. Isopropyl β-D-1-thiogalactopyranoside (IPTG)
4. E.coli culture

PROCEDURE:

DAY 1:

M9 broth and E. coli culture were prepared.

DAY 2:

To ensure sterility, the following steps were performed in the Laminar Air Flow hood.

1. Transfer 1 ml of M9 broth to two sterilized Eppendorf tubes.

2. Transfer 20μl of E. coli culture to both the Eppendorf tubes.
3. Add 50μl of IPTG and 20μl of X-gal to one of the tubes and label it as IPTG.
4. Add only 20μl of X-gal to the other tube and label it as X-gal.
5. Incubate both the tubes at 37°C for 24-48 hours in an incubator.

OBSERVATION:

The Eppendorf tube that had only X-gal was observed to be white and the tube that had both IPTG and X-gal
appeared blue.

RESULT:

Gene regulation in E.coli culture was successfully observed by visual screening method.

DISCUSSION

Understanding the Lac operon is essential to researching gene control in E. coli through visual screening
techniques. Unwanted gene expression, inconsistent outcomes, and contamination are challenges. It is essential
to ensure precise cloning, employ the right sterile procedures, and use the right controls. By finding regulating
components and mechanisms, this approach advances synthetic biology and biotechnology.

CONCLUSIONS

To better understand the function and regulation of genes, visual screening for gene regulation in E. coli is a
useful technique. The significance of the approach is demonstrated by its applications in biotechnology and
medicine. Overcoming obstacles such as contamination and variable gene expression can improve the accuracy
of findings, supporting the investigation of gene regulatory processes.
 ISOLATION OF RNA FROM LEAF SAMPLE
AIM:

To isolate RNA from Betel leaves.

PRINCIPLE:

The AMES method with TE buffer isolates RNA from plant leaves by disrupting cell membranes, denaturing
proteins, and selectively precipitating RNA while preserving its integrity using the TE buffer. Grinding the leaf
sample in AMES Buffer helps break down cell walls and release the cellular components, including RNA.
Incubation activates the enzymes that may be involved in RNA extraction or facilitating cellular component
breakdown. Chloroform is used to separate the aqueous and organic phases in the mixture. As a polar molecule,
RNA stays in the aqueous layer, while other components may partition into the organic layer. Adding NaCl and
ethanol helps to precipitate the RNA from the aqueous phase. This is because RNA is less soluble in ethanol and
the high salt concentration helps with RNA precipitation. Air-drying removes any remaining ethanol, and the
RNA is dissolved in TE Buffer which provides a suitable environment for RNA stability.

MATERIALS REQUIRED:

REAGENTS:

1. AMES Buffer
● Sodium Chloride - 1.17 g
● Magnesium Chloride - 0.04g
● Sodium acetate - 0.8g
● SDS (3) - 0.6g
● Ethanol (20%) - 4ml
● Distilled water - 16 mL
2. 5M NaCl - 20mL
3. TE Buffer
● 10mM Tris-HCl
● 1mM EDTA
4. Chloroform and isoamyl alcohol

OTHER REQUIREMENTS:

Mortar and pestle, micropipettes, centrifuge tubes, measuring cylinder, Betel leaves

PROCEDURE:

1. Weigh about 1g of betel leaf and homogenize it in 5mL of AMES Buffer using a mortar and pestle.
2. The mixture was transferred into a centrifuge tube and incubated at 37°C for 30 mins followed by adding
equal volumes of Chloroform and isoamyl alcohol (24:1).
3. Vortex the mixture and store it in ice-cold conditions (4°C) for 10 mins.
4. Centrifuge at 6000 rpm for 10 mins at 4°C. Total RNA will remain at the upper (aqueous) layer.
5. Transfer the upper layer to another centrifuge tube, and precipitate using 1/10th volume of 5M NaCl.
 6. Add double the volume of chilled ethanol.
7. Centrifuge the above mixture at 10000 rpm for 20 mins at 4°C.
8. Remove the supernatant, and collect the pellet.
9. The pellet obtained is air-dried and dissolved in 10mL of TE Buffer and stored at 4°C for further use.

RESULT:

RNA was successfully isolated from a Betel leaf sample.

DISCUSSION

RNA isolation from plant leaves is crucial for studies on gene expression. The method involves stages like
rupturing cell membranes and precipitating RNA. Contamination and RNA degradation are possible problems
Important steps include homogenization, grinding, and careful handling. Ensuring RNase-free surroundings and
proper reagent handling are crucial. RNA separation is helpful in RNA sequencing and gene expression
research, and it provides insights into biological processes.

CONCLUSION

In order to conduct gene expression investigations for research and diagnostic purposes, successful RNA
isolation is essential. High-quality RNA is ensured by overcoming obstacles such RNA degradation and
contamination using precise method. By enabling precise RNA sequencing and gene expression analysis, this
advances our knowledge of molecular biology.
 POLYMERASE CHAIN REACTION (PCR)

AIM:

To amplify a specific DNA fragment using the Polymerase Chain Reaction (PCR) technique.

PRINCIPLE:

PCR is a molecular biology technique used to exponentially amplify a specific DNA sequence. It relies on a
DNA polymerase enzyme (usually Taq polymerase) to replicate the DNA. The reaction mixture contains the
DNA template, DNA primers that anneal to the target region, nucleotides (dNTPs) to build the new DNA
strands, and buffer components to provide optimal conditions for the polymerase enzyme.The buffer helps in
maintaining the pH of reaction mixture and supplies K⁺ or NH₄⁺ for optimal enzyme activity. A series of
temperature cycles are involved for amplification of the targeted DNA.The cycling process is divided into three
main stages:Denaturation, Annealing and Extension.

Repeating these cycles leads to the exponential amplification of the target DNA.

MATERIALS AND REAGENTS:

1. DNA template
2. Forward and reverse primers
3. Taq DNA polymerase
4. dNTPs (deoxynucleotide triphosphates)
5. PCR buffer
6. MgCl2 (if not in the buffer)
7. Nuclease-free water
8. Thermal cycler
9. PCR tubes
10. Micropipettes and tips

PROCEDURE:

1. Prepared the Reaction mix by adding 16µg of Master mix (contains Buffers, Cofactors, dNTPs, Taq
Polymerase, and Copper), 2µl of DNA, and 2µl of forward and backward primers in a clean PCR tube.
2. Label the PCR tube.
3. Place the PCR tube in the thermal cycler.
4. The cycling process is divided into three main stages viz.:

i) Denaturation: separation of the DNA strands (95-98°C) for 7 minutes.

 ii) Annealing: binding of the primers to the DNA templates providing a starting point for polymerase
enzyme (50-56°C) for 2 minutes.

iii) Extension: synthesis of new DNA (72-75°C) for 5 minutes.

5. The Annealing stage was set to repeat 20 times.

6. The thermal cycler was started to execute the programmed cycling conditions.
7. The PCR was completed and the results were analyzed using Agarose Gel Electrophoresis.

RESULT:

The DNA fragment of interest was successfully amplified and analyzed using Agarose Gel Electrophoresis.

DISCUSSION

PCR is used to amplify specific DNA sequences. Two major obstacles are primer design problems and non-
specific amplification. Magnesium ion concentration, annealing temperature, and primer specificity are
important variables that impact PCR. It is essential to maintain ideal circumstances and utilize reagents
correctly. Applications of PCR in molecular biology include forensic analysis, gene cloning, and sequencing.
These demonstrate the variety and significance of PCR.

CONCLUSIONS

In molecular biology, PCR is an important method that allows for the amplification of particular DNA for a
variety of uses. The effectiveness is increased when problems such as non-specific amplification are addressed
by meticulous primer design and optimization. The application of PCR in forensic investigation and gene
cloning highlights the technology's importance in improving genetic research and diagnostics.
 AGAROSE GEL ELECTROPHORESIS
AIM:

The main aim of agarose gel electrophoresis is to separate and analyze DNA (or RNA) fragments based on their
size. It helps in identifying, quantifying, and purifying nucleic acids.

PRINCIPLE:

Agarose Gel Electrophoresis uses a gel matrix called agarose gel that is used as a medium. It is a porous matrix
that allows DNA fragments to pass through when an electric field is applied. DNA fragments are negatively
charged due to their phosphate backbone. When an electric current is applied, it moves toward the po sitive
electrode (anode). Smaller DNA fragments move faster and travel further through the gel than larger ones because
they can navigate the pores of the gel more easily. Thus, you can determine the approximate length of a DNA
fragment by running it alongside a DNA ladder (a collection of DNA fragments of known lengths). After the run,
DNA fragments are usually stained with a dye (like ethidium bromide) that fluoresces under UV light, allowing
the bands to be visualized.

MATERIALS REQUIRED:

EQUIPMENT:

Casting tray, well combs, Battery (voltage source), Gel box, Heating Mantle, Electrophoresis chamber, Gel
casting trays, Combs, Micropipette, Micropipette tips, DNA samples (25μl) and UV transilluminator.

REAGENTS:

1. TBE Buffer:

10.17g of Tris HCl, 4.95g of Boric acid, and 0.81g of EDTA are dissolved in 900 ml of water and the
pH is adjusted to 8.2

2. Agarose:

2% Agarose i.e. 0.5g of agarose is added to 25 ml of the TBE buffer. Do not dissolve the agarose before
setting the pH, and use a heating mantle to digest the agarose.

3. Ethidium Bromide (EtBr):

10µg

4. Sample preparation:

15µl of sample and 5µl of agarose gel loading dye

PROCEDURE:

1. Using tape, set the casting tray, using distilled water, and make sure the tape is put properly. No water
should leak out of the tray.
2. The combs are then fixed.
3. 0.5g of agarose powder is dissolved in 25 ml of TBE buffer by placing it on a heating mantle for 2-4
minutes without disturbing the mixture.
4. 10µg of EtBr was added to the casting tray with the aid of a micropipette
5. Pour the agarose mixture onto the casting tray (with combs) slowly to avoid air bubbles.
6. In case of tiny air bubbles, push them towards the comb with a micropipette tip.
7. Let it set at room temperature until it completely solidifies.
8. Once solidified, place the agarose gel into the gel box (electrophoresis unit) after removing the combs.
9. Add loading buffer to your DNA sample
10. Fill the gel box with the TBE buffer until the gel is covered.

11. Load your samples into the wells of the gel carefully.

12. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. (Note- A
typical run time is about 1-1.5 hours, depending on the gel concentration and voltage).

13. Turn OFF the power, disconnect the electrodes from the power source, and then carefully remove the gel
from the gel box.
14. Using a UV transilluminator, visualize the DNA bands for further analysis.

OBSERVATION:

DNA bands advanced lengthwise through the gel in an extended configuration.

RESULT:

DNA fragments were successfully separated from the sample.

DISCUSSION

A frequently used method for size-based DNA fragment separation is agarose gel electrophoresis. the issues
encountered during the testing with uneven band migration and smearing, which were typically caused by
improper voltage settings or gel concentration. Precautions include making sure that the electrophoresis settings
are correct, loading the gel precisely, and preparing the gel consistently are crucial. This approach is useful in
molecular biology for estimating size, assessing purity, and verifying PCR results when it comes to DNA
analysis. For uses like cloning, genetic research, and diagnosis, it is a vital tool.

CONCLUSIONS

DNA fragment size and integrity can be determined with great accuracy using the dependable technique of
agarose gel electrophoresis. Careful optimization can produce results that are comprehensible even in the face
of difficulties like uneven band migration and smearing. It is a crucial approach in molecular biology, as
demonstrated by its uses in genetic research and diagnostics.
 ISOLATION OF GENOMIC DNA FROM PROKARYOTIC SOURCE
(BACILLUS CEREUS OR PSEUDOMONAS AERUGINOSA)

AIM: To isolate the genomic DNA from prokaryotes (Bacillus cereus or Pseudomonas aeruginosa).

PRINCIPLE:

The methods of DNA isolation involve breakage and lysis of cells to release nuclei followed by breakage of the
nucleus to release the chromatin. Isolation of genomic DNA of bacteria involves culturing of Prokaryotes in a
culture broth ( L B broth), followed by lysis of cells using lysis buffers. The buffer is used to maintain the pH of
the solution and solubilize DNA while protecting nuclear acid from enzymatic lysis. Sodium acetate is added to
help precipitate DNA and remove impurities. Ice-cold ethanol is used to pull water from DNA molecules, so it
can precipitate well and become visible as a white cottony mass.

MATERIALS REQUIRED:

1. LB Broth (pH-7.0)
● Tryptone - 0.5g
● NaCl - 0.5g
● Yeast Extract - 0.3g
● Distilled water - 1000ml

2. Lysis Buffer (pH-5.8)

● Tris HCl - 0.1576g
● EDTA - 0.148g
● SDS - 0.25g
● Distilled Water - 20ml
3. 5M Sodium Acetate
4. Ice - Cold Ethanol
5. TE Buffer
6. Centrifuge and Rotar

PROCEDURE:

DAY 1

1. 50 ml of LB broth was prepared and autoclaved at 121℃ for 15 minutes.

2. Organism(Bacillus cereus or Pseudomonas aeruginosa) was incubated and incubated at 37℃

DAY 2

1. In an Eppendorf tube, approximately 1 ml of culture was centrifuged at 6000 rpm for 10 minutes.
2. Discard the supernatant and add 500µl of lysis buffer to the eppendorf tube.
3. Incubate at 65 degrees C for 1 hour and allow it to reach room temperature.
 4. Add 200µl of 5M Sodium Acetate to the eppendorf tube.
5. Incubate at ice cold condition for 10 minutes and centrifuge for 10 minutes at 10,000 rpm.
6. The supernatant was collected and 2 volumes of ice-cold Ethanol were added(i.e. double the volume).
7. Again incubate at ice cold condition for 30 minutes and centrifuge at 12000 rpm for 10 minutes.
8. Discard the supernatant, dry the pellet, and dissolve in 50µl in TE buffer (for further use).

OBSERVATION AND RESULTS:

The genomic DNA was isolated as white cottony thread-like fibres.

DISCUSSION

In order to release the genomic DNA, the cell wall and membrane of prokaryotic sources such as Bacillus
cereus or Pseudomonas aeruginosa must be broken. RNA or protein contamination and insufficient cell lysis are
frequent problems. Using fresh cultures, maximizing lysis conditions, and making sure all purification
procedures are followed are crucial safety measures. Applications for this technique can be found in
phylogenetic research, genetic analysis, and the creation of molecular diagnostics. Resolving these issues
guarantees high-quality DNA, which is necessary for PCR and sequencing applications later on.

CONCLUSION

Accurate genetic analysis and molecular biology studies depend on the isolation of high-quality genomic DNA
from prokaryotic sources. Using optimal techniques to overcome problems like partial lysis and contamination
guarantees the integrity and purity of the DNA. The method is essential to understanding bacterial genetics and
creating molecular tools because of its numerous uses in research and diagnostics.