HALİÇ UNIVERSITY

FACULTY OF SCIENCE AND LITERATURE

DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

2024-2025 ACADEMIC YEAR FALL SEMESTER

MOLECULAR BIOLOGY APPLICATIONS LABORATORY-I (MBG405-1)

Assist. Prof. Deniz KANCA DEMİRCİ

Laboratory Assistants
Hatice KURNAZ
Şafak ŞENER

Ymen Mazhoud (21029980113)

Group members: Noura Portio Traore, Rayan Khalil, Wael Ibrahim
Experiment date: 11/10/2024
Submission date: 18/10/2024
1. Aim:
This experiment aims to isolate genomic DNA from bacteria using manual DNA isolation
techniques that involve cell wall shearing and DNA purification.

2. Introduction:
considered one of the most abundant and simple forms of life in our environment,
bacteria play a main role in several domains such as biotechnology. Bacteria are
prokaryotic organisms, with no membrane-bound DNA (no nucleus). Their circular DNA
is found in the nucleoid. According to the cell wall structure, they are classified into two
major groups: gram-negative and gram-positive bacteria. Hans Christian Gram
discovered these two classes after developing gram staining in 1884 [1]. Gram staining
helps identify the class of bacteria, in addition to that, it helps understand their
pathogenicity, resistance, and physiology [2].

Gram-positive bacteria possess thicker peptidoglycan cell walls, the thick cell walls can
retain the crystal violet dye used in Gram staining. The bacteria appear purple when
observed under a microscope. This thick layer protects the bacteria from environmental
stress and provides structural support. Gram-negative bacteria have a much thinner
peptidoglycan cell wall but an additional outer membrane composed of
lipopolysaccharides and proteins. The additional outer membrane stops many antibiotics
from entering the cell and contributes to the pink color observed after Gram staining due
to the counterstain, safranin [3]. These structural differences affect their staining
properties and are also significant for their susceptibility to antibiotics, virulence, and o
pathogenicity [4].

The isolation of bacterial DNA is a crucial process in molecular biology, it is used for
various applications such as gene cloning, sequencing, and PCR. The method of DNA
isolation changes depending on whether the bacteria are Gram-positive or Gram-
negative. Gram-negative bacteria are easily lysed using standard protocols due to their
relatively thin peptidoglycan layer. The outer membrane can be easily sheared with
detergents like SDS, a chaotropic agent that denatures the proteins and lipids, allowing
the release of intracellular components, including DNA [5]. On the other hand, Gram-
positive bacteria require intensive lysis techniques due to their thick peptidoglycan layer.
This layer must first be broken down using enzymes such as lysozyme, this enzyme
targets peptidoglycan in a specific manner before SDS can be applied to disrupt the cell
membrane [6].

After the cell wall lysis, the bacterial DNA must be separated from other cellular
components such as proteins, lipids, and RNA. This can be achieved through a series of
chemical treatments and centrifugation. Phenol and chloroform, two organic solvents, are
often used to separate proteins from DNA. Phenol denatures proteins, while chloroform
helps in separating the organic phase from the aqueous phase where the DNA can be
found [7]. Other chemicals like sodium chloride (NaCl) are used to stabilize the DNA and

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can ensure the successful removal of residual proteins. Finally, ethanol or isopropanol is
used to precipitate the DNA out of the solution, a step that ensures the DNA is
concentrated and free from contaminants [8].

In addition to chromosomal DNA, some bacteria carry extrachromosomal DNA in the

form of plasmids. Plasmids are small, circular DNA molecules found in the cytoplasm
that replicate independently of the bacterial chromosome. Plasmids carry genes that
provide advantageous traits to bacteria, such as antibiotic resistance [9]. The ability to
isolate both chromosomal and plasmid DNA is important for various genetic studies and
applications [10]. Figure 1. demonstrates the main differences between gram-positive and
gram-negative bacteria

Figure 1. Gram-positive cell wall compared with Gram-negative cell wall [13]

DNA obtained from bacteria can be used for various molecular biology techniques.
Polymerase chain reaction (PCR) for example, where low DNA content is amplified to
generate sufficient quantities for further analysis. Restriction fragment length
polymorphism (RFLP) is another technique that relies on isolated DNA to detect
variations in DNA sequences, useful in genetic mapping, forensic analysis, and the study
of evolutionary relationships [11]. To ensure the accuracy and reliability of these
downstream applications, high-quality DNA must be obtained from these downstream
applications.

In conclusion, according to the structural and biochemical differences between Gram-

positive and Gram-negative bacteria, appropriate methods for bacterial DNA isolation
must be used. Gram-negative bacteria are more easily lysed due to their thinner cell
walls, while Gram-positive bacteria require harsher lysis techniques like enzymatic
treatments to break down their thick peptidoglycan layer. The isolation of high-purity
DNA is crucial for a variety of molecular biology techniques, including cloning, PCR,
and electrophoresis, which is why DNA extraction is a foundational procedure in

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 bacterial research [12].

3. Materials [5]:
Equipment:
- Test tube rack
- 1.5 mL Eppendorf tubes
- Micropipettes
- Micropipette tips
- CD marker
- Electrophoresis setup (tank, cassette, comb)
- Ice bath
Devices:
- Centrifuge
- Spectrophotometer
- Incubator
- Power source for electrophoresis
- UV transilluminator
- Microwave
Chemicals:
- LB medium (pH 7.3):
 1% tryptone 0.5% yeast extract
 1% NaCl
- Saline-EDTA buffer (pH 8.0):
 0.15M NaCl 4
 0.5M EDTA
 10% SDS
- 5M NaCl Tris-EDTA (TE) buffer (pH 8.0):
 10mm Tris
 1mM EDTA
- 24:1 Chloroform-isoamyl alcohol
- 95% ethanol
- 0.7% agarose gel
- 6X Loading dye (Thermo Fisher Scientific)
- 1kb GeneRuler DNA Marker (Thermo Fisher Scientific)
- Ethidium bromide (10 mg/ml) (Thermo Fisher Scientific)
Organic material:
- Bacterial culture

4. Methods [5]:
1) A bacterial culture is prepared overnight, in 50 mL LB medium (28°C at 180 rpm).

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 2) two Eppendorf tubes are used, and 1mL of the culture is taken into each tube.
3) The two tubes are centrifuged for 5 minutes at 5000 rpm.
4) The supernatant is discarded from each tube, and the pellet is dissolved in 65μl of
saline EDTA buffer. Once dissolved entirely, the two tubes are mixed.
5) The combined tube is then centrifuged for 5 minutes at 5000 rpm.
6) Once again, the supernatant is discarded, and the pellet dissolved in 70 μl saline-
EDTA buffer
7) 10 μl 10% SDS is added to the tube
8) A water bath (50°C) is prepared beforehand, and the tube is placed inside for 10
minutes.
9) 20 μl of 5M NaCl is added, and the tube is flipped several times to mix the content
10) 80 μl of chloroform-isoamyl alcohol is then added to the tube.
11) The tube is rapidly shaken by hand for 10 minutes.
12) The tube is centrifuged for 3 minutes at 5000 rpm.
13) The upper (aqueous) phase with DNA is transferred into a separate tube and 150 μl of
cold 95% ethanol is added to the new tube containing the aqueous phase.
14) The tube is centrifuged for 1 minute at 5000 rpm and the supernatant is discarded.
15) The DNA is dissolved in a 1.5 ml tube with TE buffer (20μl).
16) Once the pellet dissolves entirely, 5 μl of the isolate is mixed with 1 μl of loading dye
and loaded onto a 0.7% agarose gel. The sample is run in the gel electrophoresis
(120V for 20 minutes), and a 1 kbp marker is used as a ladder. Once complete, the gel
is observed under a UV transilluminator.
17) Absorbance measurements of the sample at 230 nm, 260 nm, and 280 nm are made
and the DNA concentration in the sample is measured.
18) The remaining sample is stored at -20°C or discarded according to the need.

5. Results:
Following the abovementioned procedure, we successfully isolated prokaryotic DNA
from the bacteria sample. The concentration and purity were measured using a
spectrophotometer. The sample was run through the gel electrophoresis and an image
was obtained. The spectrophotometry results are summarized in Table 1.

Table 1. spectrophotometry results of the isolated DNA sample

Sample A260/280 A260/230 Concentration
(μg/mL)
Prokaryotic 2.102 1.471 393.6
DNA

The sample was also run through agarose gel electrophoresis for 30 minutes and the
image is seen in Figure 2.

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 Figure 2. agarose gel electrophoresis results. (from left to right: 1kb DNA marker, group
1, group 2, group 3, group 4, group 5, group 6)
6 different groups isolated their samples from the same bacterial culture, group 4 bands
belong to our group. The first DNA bands belong to genomic DNA whereas smaller
bands belong to plasmid DNA.
Fuzzy bands are observed in the 4th well belonging to our isolated sample.

6. Discussion:
This experiment aimed to isolate prokaryotic genomic DNA, analyze its integrity via
agarose gel electrophoresis, and test its purity and concentration using spectrophotometry.
While largely successful, the results contain several areas for improvement, regarding
purity, concentration, and DNA degradation.

Spectrophotometry Analysis:

The spectrophotometric results provided important information regarding the quality and
quantity of the DNA. The measured DNA sample had an A260/A280 ratio of 2.102, this
might indicate RNA contamination of the sample. A 1.8 ratio is considered ideal and
reflects pure DNA, whereas ratios higher than 2.0 often suggest RNA contamination. This
is supported by the fact that RNA has a higher absorbance ratio at this wavelength.
Treating the sample with ribonucleases could help remove any residual RNA present in
the sample.

The A260/230 ratio was measured at 1.471, which is lower than the optimal of 1.8. A
relatively low ratio indicates the presence of salts or organic solvents like phenol, these
contaminants were not washed off thoroughly during the DNA purification steps.
Residual ethanol, phenol, or chaotropic agents from the DNA extraction process could
have interfered with the accuracy of the readings. More ethanol washes could improve
this result.

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 In terms of concentration, the spectrophotometer recorded a DNA concentration of 393.6
μg/mL. This is counted as a great DNA concentration. Such a high concentration can be
applied for downstream applications like PCR or sequencing, where a sufficient DNA
template is necessary. However, the high concentration combined with the presence of
impurities (RNA, organic material) could lead to inaccurate quantification.

Gel Electrophoresis Analysis:

Agarose gel electrophoresis is used to observe and assess the integrity of DNA. Fuzzy
bands were observed in the fourth well, this could indicate several issues:

i. Degraded DNA: some nuclease activity could lead to DNA degradation. The DNA might
have undergone degradation during the extraction process, possibly due to nuclease
activity.
ii. Sheared DNA: Rough pipetting or excessive centrifugation could have caused
mechanical shearing of the DNA, resulting in fragments. Gentle Pipetting and avoiding
excessive tube mixing during extraction can help maintain DNA integrity.
iii. Residual Contaminants: the fuzzy bands can be caused by the presence of Contaminants
such as phenol or salts which affect the migration of DNA in the gel.
iv. The faint bands observed on the gel can also be caused by uneven loading or inefficient
staining. It's possible that the ethidium bromide did not bind evenly to the DNA. In future
experiments, ensuring that the loading dye is properly mixed with the DNA might help
with better results.
The smaller bands belong to plasmid DNA. Since we can see several bands, this indicates
that there are several plasmids in the sample.

Possible Errors and Limitations:

the obtained inaccuracies can be caused by several factors:

i. Pipetting Errors: Inconsistent and rough pipetting during both the DNA extraction and
gel loading steps could have introduced errors.
ii. Contamination: The presence of contaminants like phenol or ethanol could have affected
both the concentration measurements and the quality of the bands in electrophoresis.
iii. Nuclease Activity: If the sample was not protected from DNase action, this could have
led to DNA degradation, which most likely caused the smearing and fuzziness observed
in the gel.

Improvements:

several adjustments can be made to improve this experimental procedure:

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 i. RNase Treatment: in case of RNA presence, RNAse treatment is considered the best
option to purify the DNA sample.
ii. Ethanol wash: Performing additional ethanol washes helps remove residual contaminants
such as salts and organic matter.
iii. Gentle pipetting: To avoid DNA shearing.
iv. Gel Preparation: Using a higher concentration of agarose gel (e.g., 1%) might provide
better resolution. Running the gel at the appropriate voltage and time also plays an
important role in the DNA migration pattern.
In conclusion, a high DNA concentration was obtained. However several experimental errors
have room for improvement in upcoming isolation procedures.
7. References:
[1]Bauman, R. W. (2019). Microbiology with Diseases by Body System (5th ed.).
Pearson.
[2]Madigan, M.T., Martinko, J.M., Bender, K., Buckley, D., & Stahl, D. (2017).
Brock Biology of Microorganisms (14th ed.). Pearson Education.
[3]Ryan, K. J., & Ray, C. G. (2004). Sherris Medical Microbiology (4th ed.).
McGraw Hill.
[4]Todar, K. (2020). Todar's Online Textbook of Bacteriology. Retrieved from
https://www.textbookofbacteriology.net/ Accessed on 14/10/2024
[5]Demirci, D., Çolak, C., Gürel, I., & Şener, Ş., Prokaryotic DNA Isolation. (2024).
Haliç University, Faculty of Science and Literature, Department of Molecular
Biology and Genetics.
[6]Surzycki, S. (2000). General Aspects of DNA Isolation and Purification. In: Basic
Techniques in Molecular Biology. Springer Lab Manuals. Springer, Berlin,
Heidelberg. https://doi.org/10.1007/978-3-642-56968-5_1 Accessed on the
14/10/2024
[7]Sambrook, J., & Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual
(3rd ed.). Cold Spring Harbor Laboratory Press.
[8]Kumar, A., & Suresh, C. (2019). DNA Extraction Methods for Bacterial Cells.
Journal of Applied Science and Environmental Management, 23(7), 1281-1290.
https://doi.org/10.4314/jasem.v23i7.28
[9]Demirci, D., Çolak, C., Gürel, I., & Şener, Ş. (2023). Basic Principles in DNA
Isolation [Laboratory Note]. Halic University, Department of Molecular Biology
and Genetics, Istanbul, Turkey.
[10] Tortora, G. J., Funke, B. R., & Case, C. L. (2019). Microbiology: An
Introduction (13th ed.). Pearson.
[11] Mullis, K., & Faloona, F. (1987). Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335-350.
[12] Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory
Manual (3rd ed.). Cold Spring Harbor Laboratory Press.
[13] Steward, K. (2023, December 18). Gram-positive vs Gram-negative: One
common way to separate bacteria is based on the structure of bacterial cell walls.