FACULTY OF RESOURCE SCIENCE AND TECHNOLOGY

RESOURCE BIOTECHNOLOGY
STB 2163 RECOMBINANT DNA TECHNOLOGY LABORATORY REPORT
LECTURE GROUP: 2 F

PRACTICAL NO: 2
2A: MINI‐PREP ISOLATION OF DOUBLE‐STRANDED PLASMID DNA FROM BACTERIAL CULTURE

2B: RESTRICTION ANALYSIS OF PLASMID USING COMMON RESTRICTION ENZYMES &

AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS

2C: THE PREPARATION OF COMPETENT Escherichia coli CELLS

DATE OF EXPERIMENT 25 MAY 2023

GROUP MEMBERS 1. THURKESWARRY SIVALINGAM

(81388)
2. KASTURI A/P MADASAMY (79745)
3. MARIAM SOFIAH BINTI MOHD
RAHIMI (78128)
4. NUR SYAREENA BT SULAIMAN
(78425)
LAB FACILITATOR ASSOC. PROF. DR HAIRUL AZMAN
ROSLAN

LECTURER’S NAME ASSOC. PROF. DR HAIRUL AZMAN

ROSLAN

REPORT DUE DATE 14 JUNE 2023

INTRODUCTION

In genetic engineering and molecular biology research, plasmid DNA is a crucial tool
(Lezin et al., 2011). In bacteria, it is a tiny, circular DNA molecule that exists independently of the
chromosomal DNA. Plasmids frequently contain genes that provide bacteria advantages, such
resistance to certain antibiotics or the capacity to synthesize particular proteins. A key approach in
molecular biology is plasmid DNA isolation from bacterial cultures, which enables researchers to
get pure samples of the plasmid for additional examination and manipulation. A popular technique
that makes it possible to extract plasmid DNA from a small amount of bacterial culture is the mini-
prep method.

Restriction enzymes, also referred to as restriction endonucleases, are capable of

identifying particular DNA sequences and cleaving the DNA at those locations. In particular, these
enzymes are essential for the study and manipulation of DNA molecules in molecular biology
research. Restriction enzymes are employed in the context of plasmid analysis to digest the plasmid
DNA at certain recognition sites. Researchers can create distinctive DNA fragment patterns that
reveal important details about the structure and make-up of the plasmid by utilizing various
restriction enzymes. According to Green and Sambrook (2021), the digested DNA fragments are
subsequently separated and visualized according to their sizes using agarose gel electrophoresis
(AGE).

The bacterium Escherichia coli (E. coli) is frequently employed in molecular biology
research because of its quick growth, simplicity in genetic modification, and well-characterized
genetics. For many reasons, including cloning or protein development, it is frequently required to
introduce foreign DNA into E. coli cells during research. E. coli bacteria, however, naturally resist
incorporating exogenous DNA. Cells are "competent" through a series of chemical processes,
making them more accessible to DNA molecules, which allows them past this barrier. The
incorporation of foreign DNA and subsequent expression of the desired genetic information are
made possible by this transformation process (Watson & García-Nafría, 2019).
 The production of competent E. coli cells, restriction analysis, and mini-prep separation of
plasmid DNA are three crucial molecular biology research methods. They are employed in
numerous processes, including DNA sequencing, gene cloning, genetic engineering, and the study
of the structure and functioning of DNA molecules. Researchers can manipulate and study DNA
to acquire understanding of the complex mechanisms behind biological processes by learning these
approaches.

OBJECTIVES

1. To analyze the biochemical and molecular impacts of all reagents utilized in the miniprep
plasmid DNA protocol.

2. To compare the mobility of a DNA fragment and an intact plasmid when analyzed on an agarose
gel.

3. To compare the mobility of a DNA fragment and an intact plasmid.

4. To identify and differentiate between the primary conformations of uncut plasmid DNA and to
detect them on a gel.

5. To familiarize oneself with cell competence, the initial step in transformation.

MATERIALS & METHODS

EXPERIMENT 2A: MINI PREP ISOLATION OF PLASMID DNA FROM BACTERIA

USING ALKALINE LYSIS METHOD

Materials:

● Overnight bacterial cell culture containing cloned plasmids

● Eppendorf tubes
● Cold absolute ethanol
● 70% ethanol

Solution I: (keep on ice)

● 50 mM glucose
● 10 mM EDTA, PH 8
● 25 mM Tris-HCl, pH 8

Solution II:

● 0.2 N NaOH
● 1% SDS

Solution III: (keep on ice)

● 5 M KAc (Potassium acetate), glacial acetic acid

Methods:

1. The bacterial cells from a 2 mL overnight culture were harvested by transferring the culture into
a 2 mL microcentrifuge tube and centrifuging at 8000 rpm for 2 minutes at room temperature.
2. The supernatant (culture medium) was removed carefully, and the pellet was recentrifuged for
1 minute. Remove completely any traces of liquid media from the tube.

3. The cell pellet was suspended in 100 μl of Solution I by vertexing briefly for 10 seconds.

4. 200 μl of Solution II was added to the cell suspension and mixed gently by inverting the tube 5
times. The tube was left on ice to allow the lysis reaction to occur for 5 minutes. Do not exceed 5
minutes. A clear, viscous liquid will be observed.

5. 150 μl of Solution III were added and mixed by inverting the tube 10 times. A white precipitate
will be observed. Pellet the precipitate by centrifuging at 13,000 rpm for 5 minutes. Carefully
transfer the supernatant (containing plasmid DNA) into a sterile 1.5 ml microcentrifuge tube.

6. 2 volumes of cold absolute ethanol were added to the precipitate. Mix the contents gently by
inverting the tube at least 10 times.

7. Pellet the DNA by centrifuging at 13,000 rpm for 5 minutes at room temperature. The
supernatant was discarded, and the pellet was washed with 500 μl of 70% ethanol and recentrifuged
at 13,000 rpm for 2 minutes.

8. The supernatant was discarded as much as possible, and the DNA pellet was allowed to air dry
for 15 minutes at room temperature. The DNA pellets in 50 μl of TE buffer were suspended and
dissolved.

9. The plasmid DNA sample was kept and stored at -20 °C for further analysis using agarose gel
electrophoresis (AGE)

EXPERIMENT 2B: RESTRICTION ANALYSIS OF PLASMID USING COMMON

RESTRICTION ENZYMES & AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS

Materials:

10X RE Buffer
Plasmid DNA
Restriction endonuclease
Sterile ddH2O
Eppendorf tubes
Methods

1. The reaction for restriction digestion was prepared by adding the following reagents in the
order listed to an Eppendorf tube:

Sterile ddH20 z – (w+x+y) µL

10X assay buffer one-tenth of total volume (w) w µL
Plasmid DNA, x µL
Restriction enzyme*, y µL
(1-10 units per µg of plasmid DNA)
Total volume, z µL

2. *If desired, more than one enzyme can be included if both enzymes are active in the
same buffer and the same incubation temperature.

3. Note: The volume of the reaction depends on the amount and size of the DNA being
digested. Larger DNAs should be digested in larger total volumes (between 50-100 µL),
as should greater amounts of DNA.Vendor's catalogue for the chart of
enzyme activity in a range of salt concentrations was referred to choose the appropriate assay
buffer.

4.The reaction mixture was mixed well and gently pipetted.The reaction was incubated at the
appropriate temperature (typically 37C) for 1-3 hours, in a dry heater block (or oven).

5. The enzyme(s) was inactivated by heating at 65oC for 10 minutes or by phenol extraction.

6. Prior to use in further protocols such as dephosphorylation or ligation, an aliquot of the

digestion mixture should be analyzed by agarose gel electrophoresis analysis versus the
non-digested DNA and a DNA molecular size marker, if necessary. The DNA should be
gel purified or ethanol precipitated before use in the subsequent cloning experiments.

EXPERIMENT 2C: THE METHOD TO PREPARE E. coli COMPETENT CELLS.

Materials

Buffers & Solutions

Prepare TSS solution:

Autoclave individually: 85 mL LB, 10 g PEG in 5 mL autoclaved distilled water, 5 mL DMSO, 2

mL 1 M MgCl2. Allow those to cool, then combine, filter sterilize and fridge

Media

Luria Bertani (LB) for initial culture growth.

Centrifuge & Rotors

Centrifuge machine.

Special Equipment’s (for competent cells prep and bacterial transformation)

Liquid nitrogen

Polypropylene tubes

Shaking incubator (18˚C)

Water bath at 42˚C

Chilled microfuge tubes

PROCEDURE

Step 1: Growing Bacterial Cultures (Day 1)

1) A single bacterial colony (2-3 mm in diameter) was picked from a plate that has
been incubated for 16-20 hours at 37˚C.

2) The colony was transferred into 15 mL of LB broth in at 50mL flask.

3) The culture was incubated for 16‐20 hours at 37˚C with vigorous shaking (250‐
300rpm).

Step 2: Harvesting Cells and Freezing Competent Cells (Day 2)

1) A flask of 5 mL LB broth was inoculated with 1mL of culture from the method
done in Step 1 (starter culture).

2) The flask was incubated at 37˚C with moderate shaking.

3) The OD600 of all three cultures was read. It was then continued to monitor every
15 mins until the reading is at 0.5-0.6.

4) The culture vessel was transferred to ice water bath for 15 minutes.

5) 1mL of culture was transferred into 2 microfuge tubes and then the cells were
harvested by centrifugation at 10000 rpm for 3 minutes at RT.

6) The medium was poured off and the tube was dried invertedly on the paper towels
for 1 min. (vacuum aspirator was used or tapped gently on the paper towel to remove any
drops of remaining medium adhered to the walls of the centrifuge tube).

7) The cells were resuspended gently in 90uL of ice‐cold (0˚C) TSS buffer. (You can
use immediately after this step or keep in -20C)

8) The competent cells were snap frozen in liquid nitrogen (store stock at ‐80˚C).
 9) The tube of competent cells was removed from the freezer, thawed in hand and
immediately used it.

RESULTS
 1. Based on group experiment, our band produced at the lane 6, the first three DNA bands
showed was slightly faint and thin as it has a size of 750bp. Meanwhile, the fourth DNA
band showed a size of 1000bp as the DNA band was bright and thick. Our group’s DNA
band produced smeared or degraded due to the bubbles formed during the mixing.

2. From lane 9re, we able to observe 2 bands as the 1st band size is 1500bp and the 2nd band
has a size of 250, 253. Two bands may emerge on a gel during gel electrophoresis when
plasmid DNA is digested with the restriction enzyme EcoR1, which recognises and
cleaves DNA sequences.

DISCUSSION

Experiment 2A

Plasmid is extrachromosomal circular DNA molecules, different from the normal

chromosomal DNAs and present usually in eukaryotic organisms and replicate independently.
Each plasmid possesses at least one DNA sequence that acts as the origin of replication (the place
where DNA replication begins). These DNA molecules have the ability to replicate on their own
in the right host. Plasmids are particularly helpful for the aim of making identical clones of a
section of DNA due to their ability to automatically replicate. Plasmids are employed to transfer
information, such as essential genes (which may confer resistance to specific antibiotics on their
bacterial cells), from one cell to another. Plasmids are crucial for bacterial evolution and
adaptability to the environment because they contain genes that give advantageous characteristics
on the bacterial cell (Knight, 2018)
 In this experiment, Plasmid isolation steps include harvesting cells, cells lysis, precipitation
of cell debris, proteins and genomic DNA, using centrifugation technique. Alkaline lysis is a
technique used in molecular biology to isolate plasmid DNA or other cell components like proteins
by splitting open the cells. After being cultivated, the plasmid-containing bacteria are then allowed
to lyse using an alkaline lysis buffer made up of sodium dodecyl sulphate (SDS), a detergent, and
sodium hydroxide, a strong basic. The alkali denatures the proteins responsible for preserving the
structure of the cell membrane, while the detergent separates the phospholipid bilayer of the
membrane.

Plasmid isolation begins with destruction of the cellular structure to produce a lysate,
followed by separation of the plasmid from chromosomal DNA, cell debris, and other insoluble
substances. First and foremost, a lysis buffer solution comprising sodium dodecyl sulphate (SDS)
and sodium hydroxide is used to lyse bacteria. Most cells are disrupted, chromosomal and plasmid
DNA are denatured, and the lysate is then purified by centrifugation, filtration, or magnetic
clearing. Following neutralisation with potassium acetate, only the covalently closed plasmid DNA
is able to reanneal and remain solubilized. The majority of the chromosomal DNA and proteins
precipitate in a complex generated by potassium and SDS, which is removed by centrifugation.The
bacteria are resuspended in a resuspension buffer (Tris-Cl, EDTA) before being treated with
alkaline lysis buffer (200mM NaOH) to eliminate the plasmid DNA from the E. coli host cells.
The resultant lysate is neutralised by neutralisation buffer (NaOH) which also provides the ideal
environment for plasmid DNA binding to the silica membrane column. The precipitated protein,
genomic DNA, and cell debris are then pelleted by centrifugation, and the supernatant is put onto
a column.ethanol-based wash buffer (70% ethanol) is used to removes contaminants like salts,
metabolites, and soluble macromolecular biological components. Finally, using a slightly alkaline
(TE buffer) and low ionic strength conditions, pure plasmid DNA is extracted(MyBioSource,
2019)

Experiment 2B
A restriction enzyme is a protein derived from bacteria that cleaves DNA sequences at
sequence-specific places, resulting in DNA fragments with a known sequence at each end. Certain
laboratory techniques, such as genetic engineering and recombinant DNA technologies, depend
heavily on the employment of restriction enzymes(Ganguly, 2023).

Restriction analysis, also known as restriction fragment length polymorphism (RFLP)

analysis, is a technique used to study and analyze DNA fragments. It involves the use of restriction
enzymes, which are specialized proteins that can recognize specific DNA sequences and cleave
the DNA at those sites.

In this experiment,DNA is cut at precise locations that are determined by the surrounding
DNA sequence during restriction digestion, Restriction enzymes, which recognise and bind
particular DNA sequences and cleave at specific nucleotides either inside or outside of the
recognition sequence, are incubated with the target DNA molecule to perform restriction digestion.
Sticky ends (ends of a DNA molecule that finish with a nucleotide overhang) and blunt ends (ends
of a DNA molecule that end with a base pair) can both be produced as a result of restriction
digestion.Restriction digestion is commonly employed to prepare a DNA fragment for
subsequence molecular cloning because it allows segments of DNA to be stitched together like
building blocks via ligation.Plasmid DNA template, restriction endonuclease, and a buffer are the
components of a typical restriction digestion procedure.The reaction is incubated at a specified
temperature required for optimal restriction enzyme activity before being stopped by heat. Gel
electrophoresis, which separates the digestion by molecule length (based on the negative charge
of DNA molecules) in a polymer gel to which an electric field has been applied, can be used to
assess the results of a restriction digestion(Genscript, 2019).

Experiment 2C
The discovery that phage 1 and plasmid 2 DNAs could be transferred directly into bacterial
cells after a competence induction process including low-temperature treatment with calcium
chloride established the foundation for molecular cloning of recombinant DNAs. DNA
transformation has become crucial in almost every aspect of molecular genetics. Escherichia coli
has evolved into a versatile host organism for cloned genes in a variety of assays as well as for
molecular cloning of DNA(Hanahan et al., n.d.)
 Since DNA is a very hydrophilic molecule, it typically cannot penetrate through the
bacterial cell membrane. Therefore, it is necessary to train bacteria to be able to absorb DNA if
they are to be capable of internalizing the genetic material. This can be accomplished by floating
bacteria in a solution with a high calcium concentration, which creates tiny holes in the bacteria's
cells. By incubating the DNA and the capable cells together on ice, followed by a brief heat shock
that allows the bacteria to take up the DNA, extra-chromosomal DNA will be forced to enter the
cell. When bacteria have holes in their cell membranes, they are no longer stable and can easily
perish.

The conventional method for making bacteria permeable to DNA includes treating them
with calcium ions. By briefly exposing cells to an electric field, a process known as electroporation,
bacteria can also take up DNA. However, certain bacteria have the ability to naturally shifts, which
allows them to absorb DNA from their surroundings without the need for additional care. When
using the CaCl2 approach, the competence is acquired by pore-forming bacteria by floating them
in a solution with a high calcium concentration. The DNA can then be taken into the Host cell
using thermal shock treatment at 42○C for the transformation process(MYBiosource, 2019).

Interpretation of Results

Based on the result, our experiment (lane 6) produced a bright and smear band while 6re is
a lane with plasmid DNA cut with EcoR1. It appears on lane 6 but it quite blurry in lane 6re. Bright
bands on a DNA gel indicate regions where a significant amount of DNA has accumulated,
resulting in a high concentration of DNA molecules. Smear bands in DNA gel electrophoresis
appear as a continuous blur due to the presence of DNA molecules of varying sizes in the sample.
Smearing can be caused by factors such as degraded DNA resulting from improper storage or
exposure to nucleases, contaminants or impurities, non-specific amplification in PCR due to
suboptimal conditions or primers, and uneven loading of the DNA sample onto the gel.

Larger DNA molecules migrate slowly through the gel and so are found at the top of the
gel, whereas smaller DNA molecules migrate quickly through the gel and are found towards the
bottom. The migration of DNA molecules of the same sequence in different conformations is
affected by the compactness of each conformation as they move through the gel pores.
 Lane 6 was in supercoiled form while lane 6re was a digested sample. The lower band in
lane 6 was substantially brighter than the top band. It could be that more of the molecules in that
lane were in the supercoiled form than in other forms because the supercoiled shape holds dye
more effectively. In lane 6, the DNA sample was in a supercoiled form. Supercoiled DNA refers
to circular DNA molecules, such as plasmids, that have a twisted or coiled structure. In its
supercoiled form, the DNA is tightly wound upon itself, resulting in a more compact structure.
This form is commonly observed in circular DNA molecules. In lane 6re, the DNA sample was a
digested sample. This means that the DNA in this lane was treated with restriction enzymes, which
recognize specific DNA sequences and cleave the DNA at those sites. The restriction enzyme
digestion results in the cleavage of the DNA at the targeted recognition sites, generating smaller
DNA fragments.

Supercoiled DNA is the most compact, and therefore migrates the fastest, followed by
flexible linear and, lastly, relaxed circular molecules. The relatively small, compact conformation
of supercoiled DNA means that it sustains less resistance against the agarose matrix than a larger,
floppy, open conformation of relaxed circular DNA.As a result for lane 6, the band might be made
up of two types of plasmids: a supercoiled band and a nicked band. The top band was nicked and
the bottom band was supercoiled.Therefore, for the same DNA molecule in different
conformations, the supercoiled form runs faster than the relaxed, circular form. Meanwhile for
lane 6re, the top band was linear and the bottom band was supercoiled.m. Linear DNA sustains
less resistance through an agarose gel than relaxed circular DNA, but more than supercoiled DNA,
causing it to run at an intermediate rate.

In experiment 2A, double-stranded plasmid DNA was extracted from bacterial cultures by
resuspending the pellet cells in a buffer solution. The cells were resuspended in an isotonic solution
(Solution I) containing glucose, Tris, and EDTA to destabilize the cell wall and prevent plasmid
damage. Next, an alkaline solution (Solution II) containing sodium hydroxide (NaOH) and sodium
dodecyl sulphate (SDS) was added to aid in cell lysis and the denaturation of all proteins and
genomic and plasmid DNA in the solution. NAOH and SDS, a highly alkaline solution, dissolve
cell membranes and convert double- to single-stranded DNA (dsDNA to ssDNA). The material is
then neutralised with a potassium acetate (KAc) solution (Solution III), which also separates
plasmid DNA from genomic DNA (gDNA). The simpler, larger genomic DNA precipitates out of
solution, whereas the simpler, smaller plasmid DNA renatures easily. After centrifugation, plasmid
DNA remains liquid, whereas genomic DNA and precipitated proteins form a pellet. Any leftover
plasmid DNA in the supernatant can be precipitated or purified using spin filtration, phenol-
chloroform solutions, or ethanol. In order to separate the plasmid DNA, the ethanol precipitation
procedure is used as the final step. Once the plasmid DNA has precipitated, it must be rinsed with
ice-cold 70% ethanol and allowed to dry for approximately 10 minutes so that the alcohol can
evaporate.

In experiment 2B, restriction enzyme analysis and agarose gel electrophoresis (AGE)
analysis were performed on the plasmid. As a form of defense against viral infections, many
bacterial species produce restriction enzymes. Each restriction enzyme traverses the DNA
molecule in search of a specific recognition sequence. When the enzyme divides double-stranded
DNA, DNA fragments are produced. In total, 10 L of restriction digestion were placed in the
Eppendorf tube. Gel electrophoresis is then used to separate mixtures of molecules, including
DNA, RNA, and proteins, using an electrical current. In the electrophoresis buffer are present ions
that conduct electrical current. Since they are negatively charged, DNA molecules will move
towards the positive electrode. Since the solidified agarose gel matrix will have pores of different
sizes, like a sponge, the size, shape, and charge of the molecules can affect how quickly they move
through the agarose gel. Larger molecules travel at a slower velocity than smaller ones. Based on
the results, the bands in lane 6 were smeared. This may be the result of lingering contamination.
If the negative-control PCR (which lacked template DNA) reveals a PCR product or a smear, all
reagents must be replaced. To reduce cross-contamination, use pipet tips with hydrophobic filters
that are reusable. All reaction mixtures must be prepared in a location distinct from that used to
prepare DNA or analyse PCR results. In addition, human handling of samples and reagents,
environmental contamination of reagents, and the manufacturing chain for molecular biology
reagents were plasmid contamination sources (Wally et al., 2019).

In experiment 2C, competent cells undergo transformation. This method involves the
introduction of exogenous DNA into a host cell. Once inside the cell, the DNA can, among other
things, be incorporated into the genome, replicated, and used to produce proteins. To get the DNA
past the bacterial membrane(s) and cell wall and make the bacteria "competent" for DNA uptake,
transformation can be an inefficient process necessitating the use of methodological tricks.
Normally, cells are not transformation-ready, but they can be made permeable to plasmid DNA,
and transformation-competent cells are referred to as "competent." The process by which a
competent cell acquires heritable information is known as "transformation." Due to their extreme
fragility, competent cells must be handled with the utmost care for the transformation process to
be successful. It is possible to identify competent cells that contain recombinant vectors via blue
and white screening. Recombinant vector-containing cells are able to maintain viability on Luria
Bertani (LB) medium supplemented with penicillin (Inoue et al., 1990).

Numerous bacterial species are capable of spontaneously absorbing DNA from their
environment. On the other hand, Escherichia coli is the most frequently genetically modified
laboratory microbe. Several techniques can be used to circumvent E. coli's inherent incapacity to
absorb DNA and render the organism capable of doing so. Although numerous methods exist for
transforming cells, scientists most commonly use chemical (and heat shock) or electroporation
techniques. The process of making cells competent results in the formation of holes in the cell
membrane, which makes it easier for the cells to absorb DNA from their surroundings. After
completing these competency procedures, E. coli cells are ready for DNA transformation ("3 things
to know about making competent cells", 2019).

To be useful for the creation of high-complexity libraries, competent cells must satisfy two
conditions:

(i)They must be capable of frequent transformation with ligated DNA.

(ii) They must be competent even after being frozen and preserved in liquid nitrogen. The
first step we took in our search for the optimal conditions to generate competent cells was to
optimise the standard transformation buffer (TFB) (Hanahan, 1983).

The recA 1 genotype of the E. coli DH5 strain enables the stability of plasmids containing
homologous sequences. This strain may therefore be a suitable candidate for use as a host for the
production of cDNA libraries. In addition, because it lacks endonuclease I, this strain can be
transformed with large plasmids effectively. These characteristics are crucial for the construction
of full-length cDNA libraries containing more than 6-7 kb of inserts.
 Despite the fact that it could take up to two days to reach the optimal cell concentration, it
provided good competence at harvest across a broad range of cell concentrations. The optimal
density of cells was 0.5 OD600. Hanahan (1983) documented that incubation at 37°C led to a
decrease in transformation frequency and a strong dependence of competence on cell density.

Lastly, freezing enhances competence, and storing frozen competent cells enables an
understanding of their competence prior to their use in valuable transformations such as library
construction. Commonly used as a frozen cell stabiliser, DMSO performed admirably with our
competent cells. 7o was the optimal DMSO concentration for cold shock and storage of viable
cells. By freezing in liquid nitrogen (cold shock), the transformation efficiency was multiplied by
four to five.

Safety and precautions in experiments

There are a few general safety guidelines to consider when executing the
experiments,especially when involving plasmid DNA,restriction enzymes and bacteria
cells..These safety precautions needs to be followed in order to protect onerself and maintain the
integrity of the experiment.Personal Protective Equipment (PPE) needs to be wear all the time in
the lab.Always wear appropriate PPE, including laboratory coat or gown, gloves, and safety
glasses or face shield. These are protection from potential chemical splashes, biological
contamination, and accidental spills.Next, plasmid DNA isolation needs to be performed in a
properly functioning Biological Safety Cabinet to provide a sterile and contained environment.
The BSC helps minimize the risk of contamination and protects from any aerosols or biohazardous
materials.Other than that, good aseptic techniques practice is crucial throughout the procedure to
minimize the risk of contamination. This includes working near a Bunsen burner or using a laminar
flow hood when handling open tubes or plates, flame-sterilizing tools before and after use, and
avoiding unnecessary movements or talking during critical steps.All chemicals, including buffers
and solvents, must be handled carefully in terms of chemical safety. It is necessary to follow the
correct procedures for handling, storing, and disposing of chemicals, including donning gloves and
operating in an area with good ventilation.All containers and waste items must be properly labeled
for containment and garbage disposal.Put discarded pipette tips, tubes, and other biohazardous
debris in the specified biohazard containers for disposal.In case if any emergency
occurs,emergency preparation is important.Location of safety supplies including eyewash stations,
safety showers, fire extinguishers, and spill kit needs to be identified and placed in suitable
area.Also,need to ensure that the assistant have received adequate training in plasmid DNA
isolation procedures and are familiar with the unique safety protocols developed in your
laboratory. Keep up with current safety recommendations and best practices.

Conclusion

In conclusion, three essential molecular biology research techniques include producing

competent E. coli cells, performing restriction analyses, and separating plasmid DNA using a mini-
prep..In all three tests, the biochemical and molecular effects of all compounds were successfully
analyzed and determined.We may infer from the trials that particular chemicals and reagents are
needed for each experiment in order for it to succeed. From this experiment also,Plasmid DNA
was successfully isolated from bacterial cells using alkaline lysis method.Additionally,the main
distinctions between the fundamental conformations of uncut plasmid DNA were found on a gel,
and the mobility differences between a DNA fragment and a complete plasmid were
elucidated.Finally, we discovered the first steps in genetic transformation from the experiment for
growing competent E.coli cells, which can be valuable for future molecular biology and genetic
study.
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