Corinne Hrnicek
Phoenix College Stem Train program
Dr. Chapman
15 April 2025
Abstract
In Summary, The experiment started with an RNA purification kit. After that, a DNA purification kit was used to extract DNA. DNA yield and purity were tested after the methods were followed. Multiple kits and extraction methods were used—all involved taking a 100 mg sample of brittlebush. Many involved adding buffers, boiling, centrifuging, vortexing, and discarding anything not DNA. The hypothesis tested different extraction methods. The results showed that the hypothesis was supported, and each method yielded a different result. Some had too much salt. Some had not enough. Some were within the threshold of pure DNA, and some were not.
Background information
This topic was selected because it is the project stem train students are working on this semester. With research support being what it is, we look for the most efficient way to extract DNA. This topic was also selected because it is fascinating and essential for medicine, plants, biology, and science. Extracting Plant DNA is important because “It allows scientists to analyze specific genome regions that are conserved across species. Learning more about this topic is important because It forms the basis for many downstream applications in scientific research, conservation efforts, and agricultural advancements. It is important to conduct research in this area because It helps study trait inheritance, develop genetically modified organisms (GMOs), and preserve endangered plant species. It is crucial for a wide range of applications in plant biology. It helps identify interesting mutations in plant genes, which can improve or hinder their growth in specific conditions” (The DNA Connection, 2024).
What is already known about this topic is that “DNA microarray and RNA sequencing (RNA-seq) techniques are advantageous for large-scale genome-wide studies….genomics tools cannot always provide precise information about protein levels and regulation”(Neuman, 2021). Something else that we already know is that “Recent studies have shown MN patches to be a promising method for rapid DNA extraction, with applications for the detection of allergens in food matrices” (Selz, 2023). Yet another reason that the research should be further explored is that it can help detect food allergens. We also know that “in vitro repair of DNA damage (depurination sites, strand nicks and base modifications) increases the number of samples that amplify”(Stage, 2007). Interestingly, the number of samples increases when there is In vetro repair of DNA damage. In addition, while researching previous experiments relating to DNA extraction, It was found that “Arabidopsis thaliana is widely used in the research of plant molecular biology, but RNA isolation from certain tissues is difficult” (Suzuki, 2004). It is unknown why Arabidopsis is more commonly used if it is more difficult to isolate from specific tissues. I found a passage in one of the articles I cited: "Thousands of eukaryotic genes generate mature transcripts with covalently linked ends, including exonic circular RNAs and circular intronic RNAs”(Xiao, 2019). It was a fascinating article.
“It is essential but usually difficult to extract high-quality DNA from plants for molecular work since there exist a lot of polysaccharides, hydroxy benzenes, esters, and other secondary metabolites” (Jinlu, 2022). Expanding on current knowledge is essential to conducting further research and being more efficient and accurate in extracting DNA despite polysaccharides, hydroxybenzenes, esters, and other secondary metabolites.
I will research the topic by observing how the DNA extraction tool works and experimenting with the Plant DNA extraction tool. This is my first time using the machine, so observing and experimenting will be a learning experience.
“Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method”(Comparison, 2013). Even in the experiment I am working on, we have encountered issues with how expensive it would be to buy DDT vs buying a different kit and doing DNA purification instead of RNA purification. That is another important reason to research this topic further: to be more effective and cost-efficient in DNA extraction.
Another reason it is crucial to research this topic further is because there is still so much to learn. “Scientists are only beginning to fully understand how microbial ecosystems form around plants and affect their growth. The plant microbiome can contain thousands of microorganisms, especially fungi and bacteria” (Doe, 2021). If we discover how microbial exosystems form around plants, we need to understand better and further research the thousands of different types of microorganisms. “Molecular phylogenetics serves as a “tool” to understand and describe the diversity of organisms and reconstruct evolutionary relationships using molecular data such as DNA, RNA, and protein”(Hidayat, 2016). This research can have an impact on medicine, and we can use the information from DNA purification to better describe and understand the diversity of an organism. “Total DNA was extracted from fresh materials using a GeneJET Plant Genomic Purification Mini Kit”(Hidayat, 2016).
DNA extraction is “A method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components” (Gupta, 2019).
Variable table
Name
I/D/C
Symbol
Units
Description
Type of Plant
C
I
Plant species extract
Amount of plant material
C
mg
Mass of plant material before DNA extraction
DNA extraction method
I
/
#
Method of extraction
Amount of DNA extracted
D
ng/μl
Using the same formula
Hypothesis: The amount of DNA extracted varies significantly depending on the DNA extraction method.
Research Question: How does the DNA extraction method affect the yield and purity of extracted DNA?
I expect to find results that support my hypothesis. The efficiency will vary based on the method of extraction. Each DNA sample will be a little different. I expect to learn more about DNA, plant extraction, and the tools used to extract the DNA.
Materials from left to right
Scale to measure 100 mg of sample for each and to measure balance centrifuge sample.
Cutting board and scalpel to cut sample to be exactly 100 mg
Clippers and container to collect sample
Pipette to transfer buffers and H2O into microcentrifuge tubes
Yellow kit and purple kit with buffers and materials
1.5 microcentrifuge tubes for the sample and H20 to balance the centrifuge
Container with microcentrifuge tubes containing samples and H20
Instructions for multiple extraction methods
Tips for pipette
Ethanol in orange glass container
Ice bath in purple container
On the other counter to the right
Centrifuge
Distilled water
Heat block
vortex
Procedures
DNA Extraction
30 mL of ethanol was added to wash buffers 1 and 2
box on the bottle was checked to show that we had already added the ethanol on both bottles
350 mili liters of lysis buffer A was pipetted into a 1.5 mL microcentrifuge tube
About .95 g or 95 mg of fresh plant tissue Was measured out
The material was ground with a pencil with a cap into a microcentrifuge tube
The tissue powder was transferred into a 1.5 mL microcentrifuge tube that contained 350 milliliters of lysis buffer A and then vortexed for 15 seconds
50 mili liters of lysis buffer B and 29 mili liters of RNase A were added
the sample was put in a DryBlockHeater for 10 minutes at 65 degrees Celsius. The vortex was used once every minute for 15 seconds during the dry block heating
130 milliliters of precipitation solution was added and The tube was inverted twice
The microcentrifuge tube was put in an ice bath for more than 5 minutes
The sample was centrifuged at 12500 for 15 minutes
The supernate was collected and then put into a new microcentrifuge tube
The next day 400 milliliters of plant gDNA Binding solution and 400 milliliters of 96 percent ethanol were added to the supernate and mixed
Six hundred milliliters of the prepared mixture were transferred to the spin column and centrifuged at 6,000 x g for 1 minute. The flow-through solution was discarded. The remaining mixture was applied to the same column and centrifuged at 6,000 x g for 1 minute
Five hundred milliliters of wash buffer one was added to the column and centrifuged at 12,500 for 5 minutes. The collection tube was emptied, the purification column was put back into the tub, and then re-spun at 12,500 for 5 minutes.
The collection tube containing the flow-through column was discarded and transferred to a sterile 1.5 mL microcentrifuge tube.
One hundred milliliters of elution buffer was added to the center of the column membrane. It was incubated at room temperature for 5 minutes and then centrifuged at 8,000 x g for 1 minute.
A second elution step was performed using 100 milliliters of elution buffer in the same tube.
The microcentrifuge tube was put in a freezer at -20 degrees celsius until the next day when we put 2 ml into the machine that sent information to the computer on how pure the DNA is.
Boil Method With Ethanol
1. Take 100 mg of plant material and transfer to a microcentrifuge tube.
2. Then, 200 μL of 0.9% NaCl solution (or protease solution) and 100 μL of lysis buffer (or
20% Chelex ® -100 C7901, Sigma) were added and vortexed vigorously for 10 s.
3. This mixture was heated in a water bath at 95 °C for 10–20 min, vortexed for another 10
s, and then centrifuged (Microfuge ® 20R, Beckman Coulter) at 13 000 r/min for 10 min.
4. An equal volume of isopropanol was added to the supernatant upon transfer to a fresh
Eppendorf tube and centrifuged again at 13 000 r/min for 10 min.
5. The pellet was washed with 70% ethanol and centrifuged for another 10 min at 13 000
r/min.
6. Afterwards, 70 μL of nuclease free water was added to the pellet and kept at 4 °C in a
refrigerator for 20–30 min.
7. After a final centrifugation at 13 000 r/min for 5 min, the aqueous phase was collected
leaving the soft spongy pellet.
Boil Preparation (Crude DNA Extraction)
1. Take 100 mg of plant material and transfer to a microcentrifuge tube (or PCR tube) with
200ul of sterile water. Spin the culture for 10,000xg for 1 minute.
2. Remove supernatant and resuspend pellet with 200 uL Lysis Buffer and 100 uL of
protease.
3. Heat at 95C for 3 minutes.
4. After heating, place tube in microcentrifuge and spin at 12,000x
g for 2 minutes to pellet out cellular debris.
5. Transfer the supernatant (contains genomic DNA) to fresh microcentrifuge tube and keep
on ice until ready to use.
6. Store at -20C if more testing is required.
Boil Preparation Lambda
1. Take 100 mg of plant material and transfer to a microcentrifuge tube and add 1ml of
sterile water.
2. Boil at 100°C for 10 minutes.
3. Centrifuge at 3,000 RPM for 4 minutes. Transfer the supernatant to a new 1.5 mL
microcentrifuge tube and discard the pellet.
4. Add approximately the same amount (as the supernatant) of 80% Isopropyl
Alcohol. This amount is usually 1-1.5 mL.
5. Centrifuge at 10,000 RPM for 4 minutes. Discard the supernatant and keep the
pellet.
6. Add 100 µL of 95% ETOH.
7. Centrifuge at 10,000 RPM for 4 minutes. Discard the supernatant and keep the
pellet.
8. Let the pellet air dry for about 5 minutes.
9. Add 100 µL of TAE Buffer.
*NOTE: The final product can be stored in a -20°C fridge.*
Boil Preparation HotShot
1. Take 100 mg of plant material and transfer to a microcentrifuge tube.
2. 50 µl of alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA) was added and incubated at 95°C for
1 h
3. Subsequently, 50 µl of neutralization buffer (40 mM Tris-HCL) was added and pipette-mixed
incubated at 4°C for at least 30 min
4. Centrifuge the tubes at 10,000 RPM for 4 minutes and transfer the neutralized
supernatant to a new tube.
Alkaline Lysis Buffer
To 25 ml water, add:
62.5 µl of 10 N NaOH (final concentration is 25 mM.)
10.0 µl of 0.5 M disodium EDTA (final concentration is 0.2 mM, pH should be about 12
but should not have to be adjusted.)
Make fresh every one to two months. Keep solution at room temperature.
Neutralization Reagent
To 24 ml water add:
1 ml of 1 M Tris-HCl (final concentration is 40 mM, pH should be about 5 but should not
have to be adjusted.)
Keep solution at room temperature.
Make 1 M Tris-HCl with Tris hydrochloride salt.
Data
Analysis: The data shows that the yield was not pure and had too much salt.
Conclusion: Claim: The extraction method affected the yield differently each time. For example, The first time, the method made it too salty because we did not have a centrifuge that could go to 20,000 (as the instructions said), so we centrifuged the sample to 12,500 for a longer time than was indicated in the instructions.
Conclusion: Evidence: The evidence to support this claim is in the Data. The result was 1.65 when it was supposed to be 1.8 to be pure, and therefore, it was not pure and was too salty.
Conclusion: Reasoning: The body of scientific work does support the claim. As shown by the similarities in the background research, the hypothesis, and the results, one can see that they align. Despite a slight difference in the procedure that affected the results, the hypothesis was still supported that each yield was different.
Conclusion: Merit: This research is necessary because it shows the diversity of results based on methods, which is vital to know when trying this different method. Knowing which is most efficient and which will get the best DNA yield depends on what a person is looking for, and they can decide which method to use.
References
Comparison of the efficiency of some of the most usual DNA extraction methods for woody
plants in different tissues of Vitis vinifera L. (2013). OENO One. https://doi.org/10.20870/oeno-one.2013.47.4.1559
Doe explains...the plant microbiome. Energy.gov. (n.d.). (2021)
https://www.energy.gov/science/doe-explainsthe-plant-microbiome#:~:text=The%20plant%20microbiome%20is%20an,living%20parts%20of%20their%20environment
Gupta N. DNA Extraction and Polymerase Chain Reaction. J Cytol.
2019 Apr-Jun;36(2):116-117. doi: 10.4103/JOC.JOC_110_18. PMID: 30992648; PMCID: PMC6425773.
Hidayat, T., & Priyandoko, D. (2016, March 18). Molecular phylogenetic screening of Withania
somnifera relative from Indonesia based on internal transcribed spacer region. HAYATI Journal of Biosciences. https://www.sciencedirect.com/science/article/pii/S197830191630016X
Jinlu Li, Shuo Wang, Jing Yu, Ling Wang, Shiliang Zhou. (2022) A Modified CTAB Protocol for Plant
DNA Extraction. Chinese Bulletin of Botany, 2013, 48(1): 72-78. https://doi.org/10.3724/sp.j.1259.2013.00072
Neuman, H. (n.d.). (2021) Wiley Online Library | Scientific Research Articles, journals, ... Wiley Online
Library. https://onlinelibrary.wiley.com/doi/10.1111/tpj.15410
Selz, J., Adam, N. R., Magrini, C. E. M., Montandon, F. M., Buerki, S., & Maerkl, S. J.
(2023). A field‐capable rapid plant DNA extraction protocol using microneedle patches for botanical surveying and monitoring. Applications in Plant Sciences, 11(3), e11529-n/a. https://doi.org/10.1002/aps3.11529
Skage, M., & Schander, C. (2007). DNA from formalin-fixed tissue: extraction or repair?
That is the question. Marine Biology Research, 3(5), 289–295. https://doi.org/10.1080/17451000701473942
Suzuki, Y. (2004, November). (PDF) RNA isolation from siliques, dry seeds, and other tissues of
Arabidopsis thaliana. Research Gate. https://www.researchgate.net/publication/8202391_RNA_isolation_from_siliques_dry_seeds_and_other_tissues_of_Arabidopsis_thaliana
The DNA connection: Understanding the significance of plant DNA extraction. Pure
Natural Plant Extracts Manufacturer - Over 19 Years. (2024, August 14). https://www.greenskybio.com/plant_extract/the-dna-connection-understanding-the-significance-of-plant-dna-extraction.html
Xiao, M.-S., & Wilusz, J. E. (2019). An improved method for circular RNA purification using.
RNase R efficiently removes linear RNAs containing G-quadruplexes or structured 3’ ends. Nucleic Acids Research, 47(16), 8755–8769. https://doi.org/10.1093/nar/gkz576
