INDEX

SR. EXPERIMENT NAME PG DATE SIGNATURE

NO NO

1. Extraction of genomic DNA from bacteria using

commercial kit

2. Extraction and purification of DNA band from

agarose gels (gel extraction).

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EXPERIMENT NO.1 Date:

EXTRACTION OF GENOMIC DNA FROM BACTERIA USING

COMMERCIAL KIT

AIM: To extract the genomic DNA from bacteria.

[A] ISOLATION OF BACTERIAL DNA

THEORY: The purification of genomic DNA from bacterial cultures provides the basis
for downstream molecular analysis With bacterial genomic DNA extraction the multiple
active reagents are added as a sequence of proprietary solutions with vague names such as
“Nuclei Lysis Solution” or “Protein Precipitation Solution.” Though somewhat informative,
these titles offer no information surrounding the enzymes/chemicals responsible for
degradation of various cellular components.

PRINCIPLE: The isolation of genomic DNA from bacteria involves several key
principles and steps

1) Cell lysis: The first step is to break open the bacterial cells to release the genomic DNA.
This can be achieved by various methods
a. enzymatic lysis
b. heat treatment
c. mechanical disruption
Enzymatic lysis involves the use of lysozyme to weaken the bacterial cell wall,
followed by detergent or proteinase K treatment to further break down the cell
membranes and proteins.

2) Protein removal: After cell lysis, the bacterial proteins need to be removed to isolate
the genomic DNA. This is typically done by adding protein precipitation reagents,
a. sodium dodecyl sulfate (SDS)
b. phenol-chloroform
which denature and precipitate the proteins. The mixture is then centrifuged to separate
the protein-containing phase from the DNA-containing aqueous phase.

3) DNA precipitation: The genomic DNA can be precipitated from the aqueous phase by
adding a high concentration of salt,
a. sodium acetate
b. ammonium acetate,along with a chilled alcohol,
a. Isopropanol
b. ethanol.

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 This causes the DNA to become insoluble and form visible strands or clumps. The DNA
precipitate is then collected by centrifugation.

4) DNA purification: The DNA precipitate is washed with a wash solution, usually
containing ethanol, to remove residual contaminants and salt. The wash step helps to
further purify the DNA. After washing, the DNA is typically resuspended in a suitable
buffer, such as Tris-EDTA (TE) buffer, which provides a stable environment for storage
and downstream applications. If the bacterial sample contains RNA, an optional step
involves treating the DNA preparation with an RNase enzyme to degrade any RNA
molecules. This helps to ensure the purity of the isolated genomic DNA. The isolated
genomic DNA can then be quantified and used for various downstream applications,
such as PCR, DNA sequencing, restriction enzyme digestion, or other molecular
biology techniques. It is important to note that the specific protocols and reagents used
for genomic DNA isolation may vary depending on the bacterial species, the desired
DNA yield and purity, and the available laboratory resources.

REQUIREMENTS:

● Sample: DH5α
● Reagents:
1. CTAB (cetyltrimethylammonium bromide) -
2. Β-mercaptoethanol
3. Tris-HCl buffer EDTA
4. Chloroform: isoamyl (24:1)
5. Absolute alcohol
6. 70% ethanol.
7. TAE buffer (Tris, glacial acetic acid, EDTA)
8. TE buffer
● Miscellaneous: Centrifuge, Weighing balance.

PROTOCOL:

1. Take 1.5 ml of culture in an Eppendorf tube.

2. Centrifuge the tube at 10000 rpm for 10 mins
3. Discard the supernatant.
4. Add 1 ml of CTAB and a few drops of β-mercaptoethanol and add the mixture to an
Eppendorf tube. Mix well.
5. Resuspend the cells in the buffer again and incubate for 1 hr at 60 °C.
6. Centrifuge at 10000 rpm for 5 mins.
7. Collect the supernatant and discard the pellet.
8. Add 1 ml of chloroform: isoamyl into the tube (mix well).
9. Centrifuge at 10000 rpm for 5 mins.
10. Collect the aqueous layer and add 2 volumes of chilled ethanol.

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 11. Centrifuge and collect the pellet, discarding the supernatant.
12. Add 300 µl of 70% ethanol.
13. Centrifuge collects the pellet and discards the supernatant.
14. Add 300 µl of chilled ethanol.
15. Centrifuge and discard the supernatant.
16. Keep the pellet for drying.
17. Resuspend the pellet in 30 µl of TE buffer.

RESULT & CONCLUSION:

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B] AGAROSE ELECTROPHORESIS OF GENOMIC DNA FROM
BACTERIA

AIM: To extract genomic DNA from bacteria.

THEORY: The purification of genomic DNA from bacterial cultures provides the basis for
downstream molecular analysis with bacterial genomic DNA extraction the multiple active
reagents are added as a sequence of proprietary solutions with vague names such as “Nuclei
Lysis Solution” or “Protein Precipitation Solution.” Though somewhat informative, these titles
offer no information surrounding the enzymes/chemicals responsible for degradation of various
cellular components.

REQUIREMENTS:

● Sample: Extracted genomic DNA.

● Reagents:
1. Agarose- 0.8% (bacterial DNA)
2. Ladder
3. Gel-loading dye (Bromophenol blue)
4. Ethidium bromide (1 mg/ml)
5. 50X TAE buffer (Tris, glacial acetic acid, EDTA)
● Miscellaneous: Electrophoresis tank, power pack, UV Transilluminator.

PROTOCOL:

1. Weigh agarose (0.8%) for bacterial DNA and dissolve it in a 50 ml TAE buffer.
2. Digest it in the microwave till the agarose completely dissolves.
3. Cool the dissolved agarose to some extent.
4. Fix the tray in the electrophoretic apparatus.
5. Place the comb in the tray in an appropriate position.
6. Add 5 µl of 1 mg/ml ethidium bromide and mix well.
7. Pour the gel into the tray and allow it to solidify.
8. Carefully remove the comb without disturbing the wells.
9. Place the tray into the tank containing 1X TAE buffer
10. Load the samples into the well along with the ladder.
11. Plug the electrodes into the power pack and switch on the electric supply at a constant
voltage of 110 V.
12. Allow the dye to run up to 3/4th of the gel.
13. Switch the power supply.
14. Carefully remove the tray from the tank.
15. Switch on the UV lamp to analyze the gel.

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RESULT & CONCLUSION:

REFERENCES:
● https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577976/
● http://courseware.cutm.ac.in/wp-content/uploads/2023/07/TBMS11-S12.pdf

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EXPERIMENT NO 2 Date:

EXTRACTION AND PURIFICATION OF DNA BAND FROM AGAROSE

GELS (GEL EXTRACTION)

AIM: To extract DNA bands and to purify it from agarose gel.

THEORY:
Gel electrophoresis serves as an essential analytical technique in molecular biology,
functioning by exploiting the interplay between a biomolecule’s size and charge to achieve
separation. In the context of DNA analysis, this method relies on the inherent negative charge
associated with the phosphate backbone of DNA. The separation occurs within a gel matrix
typically composed of agarose, a polysaccharide that forms a three-dimensional network of
pores. Samples containing the DNA fragments of interest are carefully loaded into designated
wells created within the gel. Upon application of an electric current, the negatively charged
DNA molecules experience an electrophoretic force propelling them towards the positively
charged electrode. However, the path is not a free run. The agarose gel acts as a molecular
sieve, with the intricate network of pores posing a challenge for the migrating DNA. Smaller
fragments, due to their compact size, can navigate the pores with relative ease, encountering
minimal resistance in their movement. Conversely, larger DNA fragments become entangled
within the gel matrix to a greater extent. Imagine a bulky object attempting to navigate a
complex maze – it gets stuck more frequently compared to a smaller object. This differential
interaction with the gel matrix translates to a significant disparity in the migration rates of the
DNA fragments. The smaller fragments zip through the gel unhindered, while larger fragments
become ensnared for extended periods, ultimately resulting in a clear separation of the DNA
molecules based on their size. By visualizing the separated bands through a post-
electrophoresis staining process, scientists gain valuable insights into the size distribution and
composition of the DNA sample.
Following the separation of DNA fragments by gel electrophoresis, gel extraction becomes a
crucial step for isolating and purifying the specific DNA fragment of interest. This process
entails meticulously excising the desired band containing the target DNA from the agarose gel
matrix. Several commercially available gel extraction kits leverage the affinity of silica-based
membranes for DNA. The excised gel slice is typically incubated in a specialized buffer that
facilitates the diffusion

Factors Influencing Gel Extraction Efficiency:

Gel extraction efficiency, the successful recovery of DNA from an agarose gel, is influenced
by several critical parameters.

● Agarose Percentage: The percentage of agarose in the gel directly impacts DNA elution.
Lower percentage gels (0.7-1.0%) offer a more open pore structure. This allows for
easier diffusion of the elution buffer and facilitates the release of DNA fragments from
the gel matrix. Conversely, higher percentage gels (1.5% or above) are denser,
presenting a greater physical barrier to elution. While they provide better resolution for
smaller fragments during separation, eluting DNA becomes more challenging.

● Size of the DNA Fragment: The size of the target DNA fragment also plays a role in
extraction efficiency. Smaller DNA fragments exhibit a higher surface area to volume

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 ratio compared to larger fragments. This increased surface area allows for more
efficient interaction with the elution buffer, promoting their release from the agarose
matrix. In contrast, larger fragments have a relatively smaller surface area, making them
less accessible to the elution buffer and potentially leading to lower elution yields.

● Excision Accuracy: The precision with which the desired DNA band is excised from
the gel significantly impacts extraction efficiency. Ideally, minimal agarose
surrounding the band should be included during excision. This is because agarose itself
does not elute and its presence in the extraction can dilute the final DNA concentration.
Additionally, excess agarose can potentially interfere with downstream applications
like PCR or sequencing. Techniques like using a clean scalpel or specialized gel
extraction tools can minimize unwanted agarose co-extraction.

● Elution Buffer Composition and Incubation Time: The composition of the elution
buffer and the duration of incubation with the excised gel slice are crucial for optimal
DNA recovery. Common elution buffers typically contain a weakly alkaline solution
(around pH 7.5-8.5) like Tris-EDTA (TE) buffer. This slightly alkaline environment
promotes the disruption of non-covalent interactions between DNA and the agarose
matrix, facilitating its release. Furthermore, sufficient incubation time allows for
thorough diffusion of the elution buffer throughout the gel slice, ensuring efficient
extraction of the target DNA.

PRINCIPLE:

Agarose gel electrophoresis separates DNA fragments by size. After staining with ethidium
bromide and visualization under UV light, the desired DNA band is excised. This DNA is
purified by incubating the gel slice in a buffer that inactivates enzymes and dissolves the gel.
The released DNA binds to a silica membrane in a spin column through ionic interactions.
Washing with ethanol removes contaminants, while a low-salt buffer elutes the purified DNA.

REQUIREMENTS:
● Agarose gel containing separated DNA fragments (visualized and documented):
● UV Transilluminator
● Sterile scalpel
● Microcentrifuge tube
● Spin column
● Centrifuge

REAGENTS:
● Agarose gel
● DNA stain (e.g., ethidium bromide)
● Gel loading buffer
● Electrophoresis buffer
● Gel extraction buffer
● Wash buffer
● Elution buffer
● Ethanol

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EQUIPMENT:

Microcentrifuge: This is a vital piece of equipment for gel extraction. It is used to centrifuge
the spin columns during various steps of the protocol. Centrifugation forces the buffer solution
through the column, facilitating processes like DNA binding to the silica membrane, washing
away impurities, and eluting the purified DNA.

PROTOCOL:
● Visualize the DNA bands under UV light in the gel image. Wear a UV-resistant face
shield and gloves for protection.
● Use a sterile scalpel to carefully excise the desired DNA band from the gel. Try to
minimize the amount of excess agarose around the band.
● Weigh the gel slice in a microcentrifuge tube. Add the appropriate volume of gel
solubilization buffer based on the weight, as specified in the gel extraction kit protocol.
● Incubate the sample at 50°C, vortexing occasionally, until the gel slice has completely
dissolved.
● Add isopropanol if specified by the kit protocol and mix well.
● Transfer the sample to a spin column provided in the kit. Centrifuge to bind the DNA
to the column membrane.
● Wash the column with the provided buffer(s) to remove impurities.
● Elute the purified DNA from the column using a small volume of elution buffer or
water.
● Quantify the purified DNA using a spectrophotometer or fluorometric method to
determine the concentration and yield.

RESULT AND CONCLUSION:

REFERENCE:

https://currentprotocols.onlinelibrary.wiley.com/journal/19343647

https://www.ncbi.nlm.nih.gov/mesh?Db=mesh&Cmd=DetailsSearch&Term=%22DNA+Rest
riction+Enzymes%22%5BMeSH+Terms%5D

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