INSTITUTE OF MEDICAL SCIENCE TECHNOLOGY (MESTECH)

MOLECULAR DIAGNOSTICS TECHNOLOGY HDB 30903

LAB REPORT 1:
DNA EXTRACTION FROM PERIPHERAL
BLOOD MONONUCLEAR CELLS (PBMC)

PREPARED BY:

NIDA’UL ‘ADNIE BINTI AHMAD RIDZUAN 12213119087

AIN NUR ASHARINA BINTI ABDUL TALIB 12213119090

FATHIMAH AQILAH BINTI ISMAIL 12213119125

MURNIEHAYATIE BINTI ISMAIL 12213119057

NOR FAIZAH BINTI MOHAMAD HASAN 12213119105

 DNA Extraction From Peripheral Blood Mononuclear Cells (PBMC)

Introduction
DNA extraction is a basic technology of molecular biology to isolate or obtain high-quality DNA
from biological samples. The process involves DNA separated from proteins, membranes, and
other cellular material contained in the cell from which it is recovered. The purity and the
integrality of DNA structure are required for various applications and different researches of
gene engineering such as genetic analysis, diagnostic, medical, and forensic science. The
rapid, effective isolation and extraction of genomic DNA from peripheral blood mononuclear
cells are essential for the inspection and analysis of clinical blood since DNA is the most
commonly used material in clinical detection. Apparently, there are various methods of
extracting DNA depending on the source, age, and size of the sample including the spin-column
method, salting out method, phenol-chloroform method, magnetic beads, and DNA extraction
kit. The basic steps in DNA extraction consist of three major steps which are cell lysis, protein
denaturation and removal of proteins and contaminants, and also the precipitation of DNA by
ethanol.

Aims and Objectives

1. To isolate DNA from peripheral blood mononuclear cells (PBMC) using spin-column
methods.
2. To measure the concentration and purity of extracted DNA using an IMPLEN
nanophotometer.

Equipment, Reagents, and Materials

1. Blood (5 ml) in an EDTA Vacutainer tube
2. Microcentrifuge tube
3. QIAamp Mini spin column
4. Collection tube with filtrate
5. LabelGuard Microliter Cell
6. IMPLEN nanophotometer
7. Fluff free tip or Kimwipe
8. QIAGEN Protease (or proteinase K)
9. Buffer AL
10. Ethanol (96–100%)
 11. Buffer AW1
12. Buffer AW2
13. Buffer AE
14. Distilled water
15. Water
16. Ethanol
17. Isopropanol
18. Dry pressurized air

Methodology
DNA extraction
1. 20 µl of QIAGEN Protease (or proteinase K) was pipetted into the bottom of a 1.5 ml
microcentrifuge tube.
2. 200 µl of the sample was added to the microcentrifuge tube. 200 µl whole blood, plasma,
serum, buffy coat or body fluids, or up to 5 x 10⁶ lymphocytes were being used up in
200 µl PBS.
3. 200 µl of Buffer AL was added to the sample. The sample was being mixed by
pulse-vortexing for 15 s to ensure efficient lysis.
4. Incubated at 56 ⁰C for 10 min.
5. The 1.5 ml microcentrifuge tube was briefly centrifuged to remove drops from the inside
of the lid.
6. The 200 µl of ethanol (96 – 100%) was added to the sample, and the sample was being
mixed again by pulse-vortexing for 15 s. After mixing, a 1.5 ml microcentrifuge tube was
briefly centrifuged to remove drops from the inside of the lid.
7. The mixture from step 6 was applied carefully to the QIAamp Mini spin column (in a 2 ml
collection tube) without wetting the rim. The cap was closed and was centrifuged at
6000 x g (8000 rpm) for 1 min. The QIAamp Mini spin column was placed in a clean 2 ml
collection tube (provided) and the tube containing the filtrate was discarded.
8. The QIAamp Mini spin column was opened carefully and 500 µl of Buffer AW1 was
added without wetting the rim. The cap was closed and being centrifuged at 6000 x g
(8000 rpm) for 1 min. The QIAamp Mini spin column was placed in a clean 2 ml
collection tube (provided), and the collection tube containing the filtrate was discarded.
 9. The QIAamp Mini spin column was opened carefully and 500 µl of Buffer AW2 was
added without wetting the rim. The cap was closed and being centrifuged at full speed
(20000 x g; 14000 rpm) for 3 min.
10. The QIAamp Mini spin column was placed in a new 2 ml collection tube (not provided)
and the old collection tube with the filtrate was discarded. Centrifuged at full speed for 1
min to eliminate the chance of possible Buffer AW2 carryover.
11. The QIAamp Mini spin column was placed in a clean 1.5 ml microcentrifuge tube (not
provided) and the collection tube containing the filtrate was discarded. The QIAamp Mini
spin column was opened carefully and 200 µl Buffer AE or distilled water was added.
Incubated at room temperature (15-25 ⁰C) for 1 min and then centrifuged at 6000 x g
(8000 rpm) for 1 min.

DNA quantification
1. The LabelGuard Microliter Cell was inserted into the cell holder with the cell windows
facing the light beam of the IMPLEN nanophotometer.
2. 1 – 2 µl (0.2 mm lid) of the sample was pipetted onto the center of the measuring
window.
3. The lid was fitted for the measurement. Make sure the lid fits exactly onto positioning
supports mounted to the body of the cell. The measurement was taken.
4. The concentration and the purity of the extracted DNA were recorded.
5. The lid was taken off and the sample was retrieved with a pipette. A fluff-free tip of the
Kimwipe was cleaned well. To clean well and cell body, water, ethanol, or isopropanol
were used.
6. The sample residues were removed from the mirror by utilizing a fluff free tip or Kimwipe.
To remove residual fluffs, dry pressurized air was used if needed. The cell was ready for
the next sample.
Discussion
1. Describe the purpose of adding the following reagents to your samples:

a. Buffer AL - A cell lysis solution that breaks open cell walls and
their nuclear membranes.

b. Ethanol - To precipitate DNA from the extracted material by

removing the solvation shell surrounding the DNA and
allowing the DNA to precipitate in pellet form.

c. Proteinase K - An enzyme that catalyzes the breakdown of cellular

proteins by splitting them into smaller peptides and
amino acids. It is also used to eliminate any cell debris
and remove proteins.

2. Buffer AE is a low salt solution containing Tris-EDTA (TE). What is TE buffer and
explain the reason why you need to keep your DNA in the TE buffer. Is there any
other type of buffer/solution that we can used to replace TE buffer?
TE buffer is a buffer solution that is commonly used in molecular biology, particularly in
procedures involving DNA, cDNA, or RNA. The name "TE" is derived from its
constituents: Tris, a common pH buffer, and EDTA, a molecule that chelates cations
such as Mg2+. We need to keep the DNA in the TE buffer because it helps in solubilizing
DNA and RNA while preventing degradation. Buffer AE can be used to replace TE
buffer.

3. State the wavelength of the nanophotometer to measure the concentration of

nucleic acids.
Using the NanoDrop spectrophotometer to measure the absorbance of sample micro
volumes is currently the most practical technique to assess DNA content and purity.
During the process of DNA extraction from PBMC, the absorbance ratios which are
260/280 or 260/230 can be utilized to evaluate the purity of DNA and to detect the
presence of contaminants in the samples. To measure the concentration of nucleic acids,
the wavelength of the nanophotometer is 260 nm.
 4. Describe the principle of nucleic acid purity measurement using nanophotometer.
The QIAamp DNA Mini and QIAamp DNA Blood Mini Kits have been designed to purify
approximately 50 μg of DNA from 200 μl of buffy coat, 5 x 106 lymphocytes, or cultured
cells with a normal set of chromosomes. Therefore there will be an average of 6 μg of
total DNA (e.g., genomic, viral, mitochondrial) from 200 μl of whole human blood. The
method required the whole blood, lymphocytes, plasma, serum, and bodily fluids that
have been treated with citrate, heparin, or EDTA. Samples may be either fresh or frozen.
For greater quantities of whole blood or cultured cells, QIAamp DNA Blood Midi or Maxi
Kits are the most recommended. The QIAamp DNA Mini Kit has all of the features of the
QIAamp DNA Blood Mini Kit, as well as the ability to purify DNA from solid tissue quickly.
From 25 mg of different human tissues, up to 30 μg of DNA may be isolated on average.

QIAamp DNA Blood Mini Kits have a QIAGEN Protease. QIAGEN Protease is the best
enzyme to use with the lysis buffer included in the QIAamp DNA Blood Mini Kit,
according to extensive testing. DNase and RNase activities are nonexistent in QIAGEN
Protease. Simply notify the Technical Service Department for assistance on whether
your lysis conditions are compatible with QIAGEN Protease when using the QIAamp
DNA Blood Mini Kit for a sample that requires a modified procedure. QIAGEN Protease
activity diminishes when more than 8 mM EDTA is combined with more than 0.5% SDS.
The QIAamp DNA Mini Kit is suitable for samples that need an SDS-containing lysis
buffer or include high amounts of EDTA. Proteinase K, which is the enzyme of choice for
SDS-containing lysis buffers used during tissue procedure but also functions well in the
blood and bodily fluid. The activity of the proteinase K solution is 600 mAU/ml solution
(or 40 mAU/mg protein). This way provides the best results in QIAamp protocols.

Safety and Precaution (if any)

1. Wear gloves all the times to avoid concentration especially from DNAse enzyme.
References
1. Alejandro Monserrat García-Alegría, Iván Anduro-Corona, Cinthia Jhovanna
Pérez-Martínez, María Alba Guadalupe Corella-Madueño, María Lucila Rascón-Durán,
Humberto Astiazaran-Garcia, "Quantification of DNA through the NanoDrop
Spectrophotometer: Methodological Validation Using Standard Reference Material and
Sprague Dawley Rat and Human DNA", International Journal of Analytical Chemistry,
vol. 2020, Article ID 8896738, 9 pages, 2020. https://doi.org/10.1155/2020/8896738.

2. Chauhan, D. T. (2018, October 21). Different Types of DNA Extraction Methods.

Retrieved from Genetic Education:
https://geneticeducation.co.in/different-types-of-dna-extraction-methods/.

3. Download - qiagen - sample to insight - qiagen. (n.d.). Retrieved October 17, 2021, from
https://www.qiagen.com/cn/resources/download.aspx?id=62a200d6-faf4-469b-b50f-2b59
cf738962&lang=en.