Laboratory Exercise No.

3
Nucleic Acid Extractions

Objectives:

The purpose of nucleic acid extraction is to separate genetic material from the rest of the cells
(proteins, DNA or RNA, cell membrane, etc). Once purified, these can be studied as individual
genes, sequence the entire genome, modify sections of DNA, and more.

Learning Outcomes:

By the end of this activity, the student should be able to:

1. Outline the three (3) basic steps in nucleic acid extraction.
2. Identify the common problems in nucleic acid extraction.
3. Extract nucleic acid from a clinical specimen.

A. Simple Human DNA Extraction from Saliva

Materials:

1. Salt
2. Microcentrifuge Tube
3. Transfer Pipette
4. Pipette and Pipette Tips
5. Absolute Ethanol
6. Small Glass or Shot Glass
7. Glass Beaker
8. 0.2 PCR tube
9. Heating block

Procedures:

1. Prepare sample tubes and label with marker.

2. Prepare saline solution by mixing a pinch of salt in a small glass with water.
3. Pour the salt water into mouth and rinse vigorously for 30 – 60 seconds.
Spit the salt solution onto a new glass.
4. Transfer sample into a microcentrifuge tube.
5. Spin the sample at 4000 G for 2 minutes.
6. Discard the supernatant and save the firmly attached pellet.
7. Remix the pellet with the remaining liquid in the tube by flicking.
8. Transfer all the suspended sample into a 0.2 mL PCR tube and have it seal
with its cap.
9. Place the sample on the heating block and heat at 99 degrees Celsius for
10 min.
10. Spin the sample using minifuge for 2 minutes at 8kG.

11. Transfer 40 µL of clear supernatant into a new PCR Tube.

12. Label and store extracted DNA at -20 degrees Celsius.

B. Bacterial DNA Extraction from Gram Negative Bacteria

Materials

1. 24 Hour Pure Culture Gram Negative Bacteria

2. Isopropanol, room temperature
3. 70% Ethanol, room temperature
 4. DNA Extraction Kit (Promega A1120)
5. Phosphate Buffered Saline (PBS)
6. 1.5 mL microcentrifuge tubes
7. Pipettors and tips
8. Water batch 80, 37, 65 degrees Celsius
9. Vortex Mixer
10. Microcentrifuge
11. Timer
12. Cold Block
13. Clean Absorbent paper

Procedures

1. Add 1 mL of an overnight culture to a 1.5 mL microcentrifuge tube.

2. Centrifuge at 13000 – 16000 x gram for 2 minutes to pellet the cells.
Remove supernatant.
3. Add 1 mL PBS and repeat step 2.
4. Add 600 µL of Nuclei Lysis Solution. Resuspend cells by gentle pipetting.
5. Incubate at 80 degrees Celsius for 5 minutes to lyse cells, and cool in room
temperature.
6. Add 3 µL of RNAse solution to the cell lysate and invert tube 2 – 5 times to
mix.
7. Incubate at 37 °C for 15 – 60 minutes. Let stand and cool at room
temperature.
8. Add 200 µL of Protein Precipitation Solution to the RNAse treated cell
lysate. Vortex vigorously at high speed for 20 seconds to mix the Protein
Precipitation Solution with cell lysate.
9. Incubate the sample in ice for 5 minutes.
10. Centrifuge at 13,000 – 16,000 x g for 3 minutes.
11. Transfer the supernatant containing the DNA to a clean 1.5 mL
microcentrifuge tube containing 600 µL of room temperature isopropanol
12. Gently mix by inversion until a thread-like strands of DNA form a visible
mass.

13. Centrifuge at 13,000 to 16,000 x g for 2 minutes.

14. Carefully pour off supernatant and drain the tube on a clean absorbent
paper. Add 600 µL of room temperature 70% ethanol and gently invert the
tube several times to wash the DNA pellet.
15. Centrifuge at 13,000 to 16,000 x g for 2 minutes. Carefully aspirate the
ethanol.
16. Drain the tube on a clean absorbent paper and allow the pellet to air-dry for
10-15 minutes.
17. Add 100 µL of DNA Rehydration Solution to the tube and rehydrate the DNA
by incubating at 65°C for 1 hour. Periodically mix the solution by gently
tapping the tube. Alternatively, rehydrate the DNA by incubating the solution
overnight at room temperature or at 4°C.
18. Store DNA at 2 – 8 °C.

C. Viral RNA Extraction using Machery Nagel Nucleospin RNA Virus Kit

Materials:

1. Machery Nagel Nucleospin RNA Virus Extraction Kit

2. Pipette and Pipette Tips (1000 µL)
3. Microcentrifuge Tubes
4. Microcentrifuge Rack
 5. Viral Transport Media with Specimen Swabs

Procedure:

1. Reconstitute Needed Reagents:

a. Add 1 mL of Lysis Buffer RAV1 to carrier RNA tube, Dissolve the
RNA and transfer it back to RAV1 bottle.
b. Wash Buffer RAV3: Add indicated volume of absolute ethanol to
wash buffer concentrate (24 mL for 10 preps; 48 mL for 50 preps; 50
mL for 250 preps)
2. Add the following to a 1.5 mL microcentrifuge tube: 150 µL sample and 600
µL of RAV1 and heat at 70°C for 5 minutes.
3. Add 600 µL of ethanol to the mixture and vortex for 10 – 15 seconds.
4. Place Nucleospin RNA Virus Column in a 2 mL collecting tube, and load
700 µL of the lysed sample. Spin at 8,000 x g for 1 minute.
5. Discard flowthrough and place the Nucleospin RNA virus column into
another new 2 mL collection tube.
6. Add 500 µL Buffer RAW to the Nucleospin RNA virus column. Spin at 8,000
x g for 1 minute. Discard flowthrough.
7. Add 600 µL Buffer RAV3 to the Nucleospin RNA virus column. Spin at 8,000
x g for 1 minute. Discard flowthrough and collection tube.
8. Place Nucleospin RNA Virus Column in a 2 mL collecting tube, and load
200 Buffer RAV3. Spin for 2-5 minutes at 11,000 x g.
9. Place Nucleospin RNA Virus Column in a new sterile 1.5 mL
microcentrifuge tube. Add 50 µL of preheated RNAse free water and
incubate for 1-2 minutes. Spin for 1 minute at 11,000 x g.

GUIDE QUESTIONS.
1. Why is it important to use a 24-Hr old bacterial culture for extraction?
2. What is the component of the lysis solution and what biomolecules they are
targeting?
3. What is the purpose of the isopropanol in the extraction?
Questions for Discussion:

1. Why is it important to use a 24-Hr old bacterial culture for extraction?

Using a 24-hour old bacterial culture for extraction is important because at this
stage, the bacterial cells have had sufficient time to grow and have already reached the
logarithmic phase of growth, ensuring enough and optimal yield of DNA. Conversely, if
the culture is too young, there may not be enough DNA or visible colonies to pick up,
while if left for too long, the cells deplete nutrients, excrete toxic metabolites, and
ultimately perish, resulting in no DNA being available for extraction.

2. What is the component of the lysis solution and what biomolecules they are
targeting?

Martinez (2022) enumerated that a crucial component of a cell lysis solution is

the detergent, which disrupts the lipid bilayer of the cell membrane, facilitating the
release of target biomolecules. Non-denaturing lysis buffers typically contain non-ionic
detergents like NP-40 or Triton X-100, which preserve the tertiary protein structure.
These detergents act as surfactants, disrupting the fatty acid bilayer without denaturing
the proteins. Additionally, the lysis solution may contain additives such as
ethylenediaminetetraacetic acid (EDTA) to inhibit proteases and protect against
oxidative damage, and Tris to buffer the pH and prevent protein denaturation as proven
by Nasrollahzadehsabet et al. (2021). In cases where denaturation is required, Bhuyan
(2010) explained that denaturing detergents like sodium dodecyl sulfate (SDS) are
included. This detergent aims to separates proteins from DNA and denatures proteins,
as DNA and SDS both have negative charges.

3. What is the purpose of the alcohol in the extraction?

As revealed by the Single‐Molecule Observation of Oda et al. (2016), the

purpose of alcohol, specifically ethanol, in DNA extraction is to induce DNA
precipitation. Ethanol precipitation is a common technique used to isolate DNA
molecules from cells, with an ethanol solution of approximately 70% (v/v) being
commonly employed for this purpose. The mechanism underlying DNA precipitation
involves a decrease in ionic dissociation due to the lower dielectric constant of ethanol
compared to water. Ethanol's dielectric constant, approximately one third that of water,
makes it favorable for DNA precipitation. However, experimental protocols typically
avoid using ethanol concentrations above 70% (v/v) to prevent contamination from cell
extracts. The unique properties of highly concentrated ethanol solutions, including the
formation of clathrate-like nanostructures with alcohol molecules in surrounding water,
contribute to the precipitation of DNA. These properties result in the association of water
nanoclusters with DNA molecules, causing the DNA to decondense and become highly
solvable in ethanol solutions. Ultimately, ethanol precipitation serves as an efficient
method for isolating and purifying DNA molecules in molecular biology applications.

Conclusions:
 In conclusion, the objectives were sufficiently achieved as I appropriately done
the basic steps in nucleic acids extraction from a clinical specimen—all while keeping in
mind the common problems of it. I have also learned about he significance of using a
24-hour old bacterial culture for sufficient amount of DNA extraction, using a lysis
solution—that contains detergents like NP-40 or Triton X-100, which disrupt the lipid
bilayer of the cell membrane to release target biomolecules, alongside additives such as
EDTA to inhibit proteases, Tris to buffer pH, and denaturing detergents like SDS if
needed—while ethanol induces DNA precipitation by decreasing ionic dissociation due
to its lower dielectric constant. As such, my misconceptions have been resolved,
including, the notion that a younger fresh bacterial culture is more suitable for DNA
extraction, when in reality, using a 24-hour-old bacterial culture ensures optimal DNA
yield as cells have reached the logarithmic phase of growth, allowing for efficient
extraction.

References:

Bhuyan, A. K. (2010). On the mechanism of SDS‐induced protein

denaturation. Biopolymers: Original Research on Biomolecules, 93(2), 186-199.
Martínez, L. M. (2022). Cell lysis buffer. Sepmag. https://www.sepmag.eu/blog/cell-lysis-
buffer
Nasrollahzadehsabet, M., Esmeilzadeh, E., Shirmohammady, N., & Heidari, M. F.
(2021). The Effect of EDTA buffer and temperature on DNA extraction from
teeth for molecular forensic assessment. Annals of Military and Health Sciences
Research, 19(2).
Oda, Y., Sadakane, K., Yoshikawa, Y., Imanaka, T., Takiguchi, K., Hayashi, M., ... &
Yoshikawa, K. (2016). Highly Concentrated Ethanol Solutions: Good Solvents
for DNA as Revealed by Single‐Molecule Observation. ChemPhysChem, 17(4),
471-473.
Qiagen. (2024). Growth of bacterial cultures. QIAGEN.
https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/technology-
and-research/plasmid-resource-center/growth-of-bacterial-cultures

Laboratory Exercise No. 4

NUCLEIC ACID / PROTEIN QUANTIFICATION ASSAYS
 NABI Spectrophotometer

Objectives:

Numerous molecular assays including traditional molecular manipulations like restriction digest,
southern blotting and even polymerase chain reaction needs a reliable method to determine
precise concentration of DNA. The spectrophotometric estimation of nucleic acid concentration
is based on the fact that absorption by RNA, RNA and their constituents follows a particular
pattern.

Learning Outcomes:

By the end of this activity, the student should be able to:

1. Operate the NABI Spectrophotometer.
2. Measure the concentration of extracted nucleic acids using NABI Spectrophotometer
3. Enumerate the advantages and disadvantages of quantifying Nucleic Acid using
Spectrophotometer.

Materials:

● Template DNA
● Blank (Nuclease Free Water)
● P10 pipettor and tips
● Kim wipes
● Nabi Spectrophotometer

Procedure:

1. Proceed to Nucleic Acid Page by touching the nucleic acid button in the main page.
2. Touch and select the measurement among dsDNA, ssDNA, RNA and Oligo.
3. Insert the title of measurement.
4. Open the head of the instrument.
5. Set the blank value by gently pipetting 2 µL of nuclease free-water to the center top of
the lower pedestal (cuvette stick should be inserted during the microscale
measurement).
6. Touch read button to start the measurement.
7. Wipe out the sample from both lower and upper pedestals with a dry and lint-free
laboratory wipe after measurement.
8. Repeat steps 5-7 for samples.

GUIDE QUESTIONS:
1. Why is it important to quantify your samples prior to PCR Assay?
2. Why is blanking important?
3. Why is bleach not recommended to disinfect / clean the probe of the instrument?
Name of Student: Edjan, Vohn Archie V. Date Performed: 3/15/24
Title of Activity: Nucleic Acid / Protein Quantification Assays Date Submitted: 3/24/24

Results:
Indicate your readings in OD and concentration.
Figure 1
Amount of Deoxyribonucleic Acid Extracted

Figure 2
Amount of Ribonucleic Acid Extracted

Note. Based on the observed concentrations of DNA and RNA in Samples 1

(DNA from saliva) and 3 (Viral DNA/RNA), it can be concluded that the
isolation procedure was conducted appropriately, yielding sufficient amounts of
both nucleic acids, indicative of a successful extraction method.

Sample 1 (DNA from saliva): 4.8 ng/µL

Sample 2 (Bacterial DNA):
Sample 3 (Viral DNA/RNA): 92 ng/µL
Guide Questions:

1. Why is it important to quantify your samples prior to PCR Assay?

Quantifying DNA samples before PCR assay is crucial for several reasons
posted by Klink (2017) and Qiagen (2024). Firstly, it ensures accurate input of DNA into
experiments, reducing the risk of erroneous results due to inconsistent DNA amounts
being used. Spectrophotometry and fluorometry are commonly employed methods for
quantification, with fluorometry being more sensitive, enabling measurement of even
nanogram quantities of DNA. Secondly, quantification helps maintain consistency
across experiments by ensuring the same amount of DNA is used each time, minimizing
variability. Moreover, knowing the quantity of DNA available allows researchers to plan
experiments efficiently, avoiding the need for repetitive DNA isolation procedures.
Additionally, quantification reveals if DNA concentration is too low for desired assays,
preventing wasted resources on experiments with insufficient DNA. Furthermore, using
specific fluorescent dyes enables discrimination between DNA species, such as single-
stranded and double-stranded DNA, which is crucial for certain applications. Most
importantly, accurate quantification ensures that results are attributed solely to DNA and
not contaminated by RNA or proteins, thereby enhancing the reliability and validity of
experimental outcomes. Therefore, quantifying DNA samples before PCR assay is
imperative for ensuring experimental accuracy, reproducibility, and validity.

2. Why is blanking important?

Blanking, the process of measuring background inherent to the

spectrophotometer or solvent used, is crucial in sample preparation and analysis for
several reasons (Koeris, 2024). Firstly, blanks play a vital role in quality control and
quantitative analytical methods, serving to track contamination sources or sample
degradation. Different types of blanks, such as ambient blanks, calibration blanks, and
method blanks, are utilized to identify and correct for various sources of interference or
contamination throughout the analytical process (Rayni, 2018). For instance, in a
laboratory setting, blanks are used to trace and rectify issues like contamination from
solvents or equipment, ensuring accurate results. Secondly, blanks are essential for
establishing detection limits, including the limit of blank (LOB), limit of detection (LOD),
and limit of quantitation (LOQ), which are crucial for determining the smallest detectable
analyte amount with statistical precision. This statistical relationship between the limits
underscores the importance of blanks in accurately assessing analyte concentrations,
particularly at low levels. Moreover, blanks aid in resolving anomalies in analysis
results, as illustrated by scenarios like identifying multiplicative interferences causing
spurious peaks in chromatograms or uncovering potential causes behind outlier values
in field samples.
 3. Why is bleach not recommended to disinfect / clean the probe of the instrument?

As been proven by Harris et al. (2006), bleach is not recommended for cleaning
the probe of the instrument because it has been found to have significant detrimental
effects on the quality of DNA profiles. In a study investigating the impact of different
cleaning agents on DNA analysis, bleach exhibited the most pronounced negative
effects, particularly on porous materials. Despite attempts to obtain profiles from non-
porous materials, reliability was compromised when compared with duplicates. Bleach-
treated materials showed a continued decline in profile quality over time, indicated by
increased scatter of heterozygote imbalance (Hbx). In contrast, substrates cleaned with
soap or non-chlorine disinfectant did not exhibit this degradation. Tuccinardi (2020)
explained that exposure to left-out sodium hypochlorite (NaOCl), commonly known as
bleach, leads to the degradation of DNA via oxidative harm and the formation of
chlorinated base products. As the concentration of NaOCl rises, DNA strands undergo
cleavage, resulting in the fragmentation of the genetic material into progressively
smaller fragments.

Conclusions:

In conclusion, the objectives were sufficiently achieved as successfully operated

the NABI spectrophotometer in concentrating nucleic acids while keeping in mind some
of its limitations. I also learned in the laboratory about the accurate quantification of
DNA samples before PCR assay towards accuracy, reproducibility, and validity, while
blanking is essential for tracking contamination sources and establishing detection
limits; however, bleach is not recommended for cleaning the probe of the instrument
due to its significant detrimental effects on DNA profiles, including oxidative damage
and cleavage of DNA strands. One common misconception that has been clarified is
that bleach is an appropriate cleaning agent for DNA analysis equipment, when in fact,
its use can significantly degrade DNA profiles rather than effectively disinfecting them.
References:

Harris, K. A., Thacker, C. R., Ballard, D., & Court, D. S. (2006, April). The effect of
cleaning agents on the DNA analysis of blood stains deposited on different
substrates. In International congress series (Vol. 1288, pp. 589-591). Elsevier.
Klink, S. (2017, June 12). Six (and a Half) Reasons to Quantitate Your DNA - Promega
Connections. Promega Connections. https://www.promegaconnections.com/six-
and-a-half-reasons-to-quantitate-your-dna/
Koeris, M. (2024). Addgene. https://www.addgene.org/protocols/dna-quantification/#
Qiagen. (2024). Quantification of DNA. QIAGEN.
https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-
guide/dna/analysing-dna/quantification-of-dna
Raynie, D. E. (2018). The vital role of blanks in sample preparation. Chromatography
Online. https://www.chromatographyonline.com/view/vital-role-blanks-sample-
preparation
Tuccinardi, A. (2020). Investigating the Efficacy of DNA Damage with Bleach in
Forensic Laboratories and at Crime Scenes. University of New Haven Honors
Program 2019-2020 Honors Thesis. Retrieved from
https://digitalcommons.newhaven.edu/honorstheses