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

Experiment 3: PROKARYOTIC DNA ISOLATION WITH KIT

Ymen Mazhoud (21029980113)

Group members: Noura Portio Traore, Rayan Khalil, Wael Ibrahim
Experiment date: 18/10/2024
Submission date: 25/10/2024
1. Aim:
This experiment aims to isolate genomic DNA from bacterial cells using the Vazyme
FastPure DNA isolation Mini Kit. This experiment uses silica gel column purification
to isolate high-quality DNA.
2. Introduction:
Molecular biology has a wide array of downstream applications, including PCR, gene
cloning, sequencing, and gene expression analysis. All these applications require
DNA isolation beforehand. The purity and quality of the isolated DNA are crucial in
determining the success of these applications, contaminants such as RNA, lipids, or
proteins can significantly alter enzymatic reactions [1]. The initial DNA isolation
procedures relied on organic solvents like phenol-chloroform. This method is
hazardous due to the toxic chemicals used and time-consuming [2]. Modern
procedures such as column-based purification techniques, have made DNA isolation
more reliable and safer.

The Vazyme FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit is a

modern technique. It uses silica gel column technology. This method allows selective
DNA binding under high salt conditions while washing away any impurity [3]. The
silica matrix efficiently binds DNA while proteins and/or salts can be washed away,
giving highly pure DNA.
In prokaryotic DNA isolation, a more straightforward extraction process is undergone
due to the simplicity of the bacterial genome. Prokaryotic DNA is much simpler than
eukaryotic DNA where the chromosome lacks the complexity found in eukaryotic
DNA, this results in a more efficient and rapid extraction method [4].
during this experiment, Gram-negative bacteria were used, they are characterized by a
thicker cell wall which adds complexity to the membrane lysis step. Therefore,
enzymatic and detergent-based techniques are required to break down the cell wall
and access the DNA [5].
Ensuring that all cells in the sample undergo lysis is a critical step in DNA isolation
since any presence of intact cells can reduce the yield in a significant way. Proteinase
K can help achieve complete cellular lysis by digesting proteins that are able to
degrade/bind to DNA [6].
Using commercial kits comes with several advantages. First, they eliminate the use of
hazardous chemicals like phenol. Second, they shorten the time required to complete
the experiment. Last, the DNA quality and purity are much higher when isolated
using kits, allowing for more reliable downstream applications [8].
in addition, this kit has wide applicability for several tissue samples like blood,
cultured cells, and bacteria, making it a versatile tool [9]. Figure 1 summarizes the
DNA isolation using a kit in general.
 Figure 2. DNA isolation using a commercial kit [11]

3. Materials [8]:
Equipment:
- Micropipette
- Micropipette tips
- Eppendorf tubes
- Centrifuge tubes
- FastPure gDNA Mini Columns (Vazyme FastPure Kit)
- Tube rack
- CD marker
- Electrophoresis setup

Devices:
- Centrifuge
- Vortex
- Incubator
- Gel electrophoresis system
- Gel imaging system
- Spectrophotometer
Biological material:
- Bacterial culture (Gram-negative bacteria)
Chemicals:
- Buffer ACL
- Buffer BCL
- Buffer WA
- Buffer WB
 - Proteinase K
- Elution Buffer
- 0.7% Agarose gel
- 6X Loading dye (Thermo Fisher Scientific)
- 1kb GeneRuler DNA Marker (Thermo Fisher Scientific)
- Ethidium bromide (10 mg/ml)

4. Methods [8]:
Bacteria sample (gram-negative bacteria):
1- 1mL of bacterial culture solution (<3.0 x 10 9 bacteria) is centrifuged at 10000 rpm
for 1 min, and the supernatant is discarded.
The bacteria number is measured using a spectrophotometer. OD 600= 1.0 means
that the solution contains 1.5 x 109 /mL
This step is repeated for a second tube.
2- 100 µL of buffer ACL is first added to the pellet of each tube from step 1. The
tubes are then merged and a total volume of 200 µL must be obtained for our final
tube. 20mL of proteinase K is added to the tube. 200 mL of buffer BCL is also
added. The sample is thoroughly mixed by vortexing for 30 sec
3- The mixture is incubated at 56 ͦ C for 10 minutes, the tube is inverted every 2
minutes during the incubation period.
4 µL of RNase A can be added in case of presence of RNA residues
Column purification:
1- 150 µL of absolute ethanol is added to the sample. The tube is vortexed for 10
seconds. If some flocculent precipitate appears, the tube can be centrifuged to
collect the liquid. If no precipitate is observed, proceed to step 2
2- The FastPure gDNA Mini Columns II is placed in a 2 mL collection tube. The
mixture is transferred to the collection tube (even the precipitate). the tube is
centrifuged at 12000 rpm (13400 x g) for 1 minute.
3- The liquid is discarded from the collection tube. The column is placed back on top
of the same collection tube and 500 µl of buffer WA is added along the column
wall. The tube is centrifuged at 12,000 rpm (13,400 x g) for 1 min.
4- The flow-through is discarded for a second time and the adsorption column is
placed on top of the collection tube for a second time. 600 μl Buffer WB is added
along the tube wall (Check whether absolute ethanol has been added). The tube is
centrifuged again at 12,000 rpm (13,400 x g) for 1 min. the flow-through is
discarded
5- Step 4 is repeated one more time.
6- The adsorption column is placed in the collection tube. the empty column is
centrifuged at 12,000 rpm (13,400 g) for 2 min.
 After centrifugation, the lid is opened and left to air-dry for 5 min to completely
remove the residual ethanol.
7- The adsorption column is transferred to a new 1.5 ml centrifuge tube (Self-
prepared). 50µl of Elution Buffer is added to the center of the adsorption column
membrane and incubated at room temperature for 3 min. the tube is centrifuged at
 12,000 rpm (13,400 x g) for 1 min.

Note: The following steps can help to increase DNA yield.

 Pre-heating the Elution Buffer to 55°C before the elution step.
 Reinserting the solution from the first elution to the adsorption column when
carrying out the elution.
8- The adsorption column is discarded at last, and the DNA is stored at -20°C.

5. Results:
Using the Vazyme FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit, we
carried out the genomic DNA isolation from Gram-negative bacteria. After
completing the series of purification steps, the extracted DNA was subjected to both
spectrophotometry and 0.7% agarose gel electrophoresis to assess its concentration
and integrity.
Spectrophotometry Results:
The concentration of the isolated DNA was measured using a spectrophotometer.
Table 1. summarizes the readings obtained.
Table 1. spectrophotometry readings obtained.
Sample A260/280 A260/23 Concentration
0 (μg/mL)
Prokaryotic 5.793 9.882 16.15
DNA

Agarose Gel Electrophoresis: the purified sample was analyzed using 0.7% agarose
gel electrophoresis. The DNA was loaded alongside a 1kb DNA marker to estimate
the size of the isolated fragments. Figure 2. Is an image of the UV image of the DNA
samples.

Figure 2. Electrophoresis results (the last band belongs to our group)

6. Discussion:
This experiment aimed to isolate genomic DNA from prokaryotic cells using the
Vazyme FastPure DNA Isolation Mini Kit. The quality and purity of the isolated DNA
was assessed using spectrophotometry and Agarose Gel electrophoresis.
Unfortunately, the results were very different from the expected outcome and normal
range, raising questions about the potential mistakes made throughout the
experimental procedure. The abnormal results were seen in both assessment methods
which indicates that the isolation did not yield high-quality DNA.
Spectrophotometry Analysis
The spectrophotometric readings gave valuable insight into the purity of the isolated
DNA. The A260/280 reading was 5.793, while the A260/230 ratio was 9.882. These
values deviate from the optimal ratio value of 1.8-2.0, which is considered necessary
for high-quality and purity DNA
A260/280 Ratio: This ratio determines the presence or absence of protein
contamination in the sample. A value of ~1.8 is considered excellent for pure DNA.
Fluctuations from this value indicate protein contamination, and in our case, a very
high contamination. Our value of 5.793 is abnormally high, suggesting an unusual
contamination rate or experimental error.
A260/230 Ratio: This ratio determines organic compounds and salt contamination in
the DNA sample such as ethanol or carbohydrates. the expected value for pure DNA
ranges around 2.0–2.2. Our result of 9.882 is highly unusual and supports the
previous observation regarding the A260/280 ratio.
Concentration: The concentration was 16.15 μg/mL, this value is low compared to
the values obtained using kits. Commercial kits are known to have higher
concentrations due to the straightforward procedure. A typical range of 20–100
µg/mL is usually expected for prokaryotic DNA isolation [10]
Electrophoresis Analysis
Unlike the spectrophotometric data, the gel electrophoresis results showed a strong
band near the top of the gel, this band is at the same level as the first band from the
DNA marker, indicating the presence of large genomic DNA and no fragments
This result contrasts with the initial assumption of degraded DNA based on
spectrophotometry; therefore, we can conclude contamination but not necessarily
degradation. The absence of smaller bands towards the lower portions of the gel and
the lack of smears support our observation.
The contrasting results can be due to the incomplete Washing of the Column,
and the presence of residual Contaminants.
To improve the quality and purity of future DNA isolations, the following
adjustments can be made:
- Extending the washing steps reduces Contamination
- Enhancing Protein Digestion and ensuring that the Proteinase K is fully active.
 In conclusion, while the spectrophotometer results show great contamination, the
Agarose gel electrophoresis results show intact DNA. The commercial kit might have
been kept under inadequate conditions which resulted in the malfunction and low
separation quality that caused contamination. The experiment must be repeated using
another kit kept under better conditions.

7. References:
[1] Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual
(3rd ed.). Cold Spring Harbor Laboratory Press.
[2] Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from
micro-organisms. Journal of Molecular Biology, 3(2), 208-218.
[3] Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P. M.,
& van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic
acids. Journal of Clinical Microbiology, 28(3), 495-503.
[4] Wilson, K. (2001). Preparation of genomic DNA from bacteria. Current Protocols
in Molecular Biology, 2.4.1-2.4.5.
[5] Helinski, D. R. (1973). Replication of plasmid DNA in Escherichia coli. Annual
Review of Biochemistry, 42(1), 645-678.
[6] Malik, K., & Brown, T. A. (2005). The isolation of high-molecular-weight DNA
from bacteria. In Brown, T.A. (Ed.), Gene Cloning and DNA Analysis: An
Introduction (5th ed., pp. 35-40). Blackwell Publishing.
[7] Vazyme. (2024). FastPure DNA Isolation Kit Manual. Vazyme Biotech.
[8] Demirci, D. K. (2024). Prokaryotic DNA Isolation with Kit. Laboratory Notes,
Haliç University, Department of Molecular Biology and Genetics.
[9] Qiagen. (2020). Silica-gel-based DNA purification: Principles and methods.
Retrieved from https://www.qiagen.com/us/resources/resourcedetail?id=c8f1fcdc-
5680-4e89-9286-e82decb8e630&lang=en
[10] Qiagen. (2023). DNeasy blood & tissue handbook: DNeasy blood & tissue kit;
DNeasy 96 blood & tissue kit. Qiagen.
[11] https://www.generi-biotech.com/products/kit-for-isolation-of-dna-from-body-
fluids/ Access date: 24/10/2024.