How to Extract RNA from Swab & Saliva Samples

HOW TO EXTRACT RNA FROM SWAB AND SALIVA SAMPLES

THE BEST METHOD FOR EFFICIENT RNA EXTRACTION

COVID-19 testing has been at the forefront of everyone’s mind since the pandemic started in early 2020. But what goes into a COVID-19 test, or any respiratory virus test for that matter? What devices are used for sample collection? What are the steps involved in RNA extraction? And how have testing solutions evolved to meet the changing demands of the pandemic?

WHAT SAMPLE TYPES ARE COMMONLY TESTED FOR COVID-19?

COVID-19 tests begin with collecting a sample. Because SARS-CoV-2 is a respiratory virus, this sample is typically collected below the upper respiratory system. Infected cells of the upper respiratory tract shed viral RNA, which can then be quickly detected by reverse transcriptase polymerase chain reaction (RT-PCR) or for more specificity, by genetic sequencing.

Swab samples, collected from the upper or lower parts of the throat (the nasopharynx or oropharynx, respectively), have been considered the gold standard due to their high sensitivity.[1] These swab samples, however, must be collected by trained healthcare professionals wearing full personal protective equipment (PPE). This requirement has led to PPE shortages throughout the COVID-19 pandemic, bottlenecking mass testing efforts.

Saliva samples have emerged as an alternative to swab samples for viral diagnostics. Not only are saliva samples easy to collect — they can be self-collected in the comfort of one’s home — their sensitivity is comparable to that of swab samples. These features make saliva samples an attractive complement to swab samples.

WHAT SAMPLE TYPES ARE COMMONLY TESTED FOR COVID-19?

The next step after sample collection is RNA extraction. But how does RNA extraction work for saliva and swab samples? Does it differ from other sample types?

When using spin columns, the general RNA extraction workflow for saliva and swab samples is similar to other sample types. There are four basic steps in spin column RNA extraction:

  1. Sample lysis — a lysis buffer is added directly to the sample. In SafeCollect Collection KitsDNA/RNA Shield serves as an all-in-one sample lysis and preservation buffer. (In other words, sample lysis occurs during sample collection.) Some RNA extraction kits may include lysis tubes for mechanical lysis and enzymes (e.g. Proteinase K) for enzymatic lysis.
  2. Bind RNA – after lysis, ethanol or isopropanol is added to the sample, mixed, and then the entire mixture is added to a spin column. The addition of an alcohol helps promote RNA binding to the column’s silica matrix. RNA is brought into contact with the silica matrix by centrifugation. Optionally, DNase can be applied on-column after RNA binding (step 2) to eliminate contaminating DNA.
  3. Wash RNA — once RNA is bound, it can be washed by applying a wash buffer containing ethanol. Because the ethanol promotes RNA binding to the column’s silica matrix, RNA is retained within the column during washing/centrifugation. Additional centrifugation can be done to remove residual ethanol from the column matrix.
  4. Elute RNA — an elution buffer (typically nuclease-free water or a Tris-EDTA buffer) is applied to the column. The elution buffer hydrates the RNA, allowing it to dissociate from the column’s silica matrix. A final centrifugation step pools the purified RNA sample in the bottom of a collection tube, ready for analysis.

Alternatively, high-throughput kits use magnetic beads in place of spin columns, but the general workflow is the same. Samples are lysed, RNA is bound to the beads, the beads are washed, and RNA is finally eluted from the beads.

HOW TO CHOOSE AN RNA PURIFICATION KIT

Choosing the best RNA purification kit for your application can be daunting because there are so many available options, and it is not always clear what differences exist between kits.

Zymo Research offers a variety of extraction kits, as part of a complete workflow. From column-based to automation-friendly kits, the choice depends on your specific experimental needs. For today’s pandemic, the Quick-DNA/RNA Viral Kit is the high-throughput solution for early viral detection.

Quick-DNA/RNA Viral Kit is designed for the quick recovery of viral DNA/RNA from a variety of sample types, including saliva and swab samples. The DNA/RNA purification kit comes with DNA/RNA Shield for sample collection, virus inactivation, and nucleic acid preservation. If you collect samples using Zymo Research’s DNA/RNA Shield sample collection devices, you can proceed straight to purification. Additionally, the kit features a one-step binding buffer which aids in efficient extraction and low copy viral detection.

Optionally, to reduce the viscosity of samples — saliva can be quite viscous! — proteinase K digestion makes sample handling easier and also eliminates nucleases that could degrade the nucleic acids in your sample. A recent correspondence in the New England Journal of Medicine featured proteinase K in its comparison of saliva and swab COVID-19 testing.[2]

Enabling automation using robotic sample processor for high-throughput extraction and viral detection has proven to meet the growing demands of routine application, especially during the COVID-19 pandemic.

ENABLING MASS TESTING WITH AUTOMATION

The kit described above works well for routine applications in which a handful of samples need to be processed at once. But mass testing, where samples number in the hundreds to thousands, requires a more scalable approach.

Magnetic beads can be used instead of spin columns to increase the throughput of viral DNA/RNA extraction. The Quick-DNA/RNA Viral MagBead Kit is a scalable and automatable magnetic bead format. The DNA/RNA purification kit can be used to process hundreds of reactions on any robotic liquid handler or bead mover. For example, 96 preps can be processed in just 22 minutes when using an automated system.

When paired with automation-friendly SafeCollect devices and the Quick SARS-CoV-2 rRT-PCR Kit, the Quick-DNA/RNA Viral MagBead Kit can be used for automated extraction and detection of SARS-CoV-2 viral RNA in saliva and swab samples from start to finish.

Whether you are testing swabs or saliva, a few samples or thousands, Zymo Research offers solutions supporting every step of the process, from sample collection to RNA extraction and beyond.

REFERENCES

Single Domain Antibodies

Single Domain Antibodies: Therapeutic Tools for the Future?

The Advantages of Single Domain Antibodies

Conventional antibodies such as monoclonal (mAb) and polyclonal (pAb) antibodies have been at the forefront of biomedical research, use in diagnostic assays therapies against cancer, immune disorders, and infectious diseases. The market for antibodies is growing significantly as the need for these tools is ever-increasing to keep up with the constant battle between diseases and human health.

Disadvantages of Conventional Antibodies

Although conventional antibodies serve as the foundation for highly successful research, diagnostic and therapeutic tools, they do have their disadvantages such as stability over a narrow pH and temperature range and may not be able to access particular active sites on proteins.

These disadvantages might not hinder your research results, but a smaller-sized single domain antibody can increase therapeutic efficacy. A study from the Annals of Medicine, about the challenges in monoclonal antibody-based therapies, pointed out how the current manufacturing and purification processes of monoclonal antibodies cause limitations in the production capacity of therapeutic antibodies, which leads to an increase in cost (1). In a study by Vanlandschoot et al. (2), the advantages of sdAbs were reviewed in relation to their possible therapeutic applications against various viral diseases such as human immunodeficiency virus-1 (HIV-1), influenza A virus, reparatory syncytial virus (RSV), are discussed. View here. Such studies culminated in the first FDA-approved sdAb against von Willebrand factor to treat the blood disease Acquired Thrombotic Thrombocytopenia (3).

How Single Domain Antibodies Can Help

Single domain antibodies from camelid, aim to be the cutting-edge tool for antibody research in cellular mechanisms, cancer, and infectious diseases. Single domain antibodies lack light chains and are smaller and more stable than conventional antibodies yet they possess a fully functional antigen-binding capacity. Due to their size (approximately 15 kDa) and their longer and structurally unique Complementarity Determining Region 3 (CDR3 region), a single domain antibody is adept at reaching otherwise inaccessible unique conformational features on a target that may play a crucial role in the molecular mechanisms of disease.

Features and Benefits of Single Domain Antibodies

Here are ways single domain antibodies can help excel your research:

  • Smallest functional antibody unit at ~15kDa; conventional antibody is ~150kDa
  • Enhanced tissue penetration, can cross the blood-brain barrier
  • Unique binding capacity to small cavities or clefts
  • High affinity and specificity
  • Highly stable at room temperature and under extreme temperatures and pH
  • High solubility, great imaging agents due to rapid clearance in vivo
  • Cost-effective, large-scale production

The unique properties of size, stability and solubility for a single domain antibody allow breakthroughs in the field of cancer research, drug development, and therapy. With a variety of ways to use single domain antibodies and the ability to effectively target cancer cells, it’s no surprise that single domain antibodies are on the front lines in the fight against cancer.

Enhance your Research with ProSci

ProSci offers Single Domain Antibody Services from Immunization to Production. Throughout all six phases (from immunization to production), single domain integrity is ensured with various milestones and an unwavering commitment to customer satisfaction. If your application calls for single domain antibodies, purchase ProSci single domain antibodies with confidence.

The ProSci sdAb prototype has a myc-tag for easy detection by an anti-myc antibody.

ProSci provides sdAbs against immune checkpoint targets such as PD-L1, PD-1, LAG3, and TIGIT. Explore the range of ProSci Immune Checkpoint Single Domain Antibodies.

At the onset of the COVID-19 pandemic, ProSci developed antibodies against the S1 and S2 domains as well as trimers of the SARS-CoV-2 virus wildtype and of several Variants of Concern. Explore the full range of SARS-CoV-2 Single Domain Antibodies.

References

  1. Samaranayake, H., Wirth, T., Schenkwein, D., Räty, J. K., & Ylä-Herttuala, S. (2009). Challenges in monoclonal antibody-based therapies. In Annals of Medicine (Vol. 41, Issue 5, pp. 322–331). Informa UK Limited. https://doi.org/10.1080/07853890802698842
  2. Vanlandschoot, P., Stortelers, C., Beirnaert, E., Ibañez, L. I., Schepens, B., Depla, E., & Saelens, X. (2011). Nanobodies®: New ammunition to battle viruses. In Antiviral Research (Vol. 92, Issue 3, pp. 389–407). Elsevier BV. https://doi.org/10.1016/j.antiviral.2011.09.002
  3. Scully, M., Cataland, S.R.,  Peyvandi, F., Coppo, P., Knöbl, P., Kremer Hovinga, J.A., Metjian, A., de la Rubia, J., Pavenski, K., Callewaert, F., Biswas, D., De Winter, H. and Zeldin, R.K. for the HERCULES Investigators. (2020). Caplacizumab Treatment for Acquired Thrombotic Thrombocytopenic Purpura. https://pubmed.ncbi.nlm.nih.gov/30625070/

Scaffolds Library

Scaffolds Library

To meet customers’ drug discovery needs better, InterBioScreen Ltd. offers you the opportunity to obtain compounds synthesized around the Scaffolds from InterBioScreen’s libraries.

• Select from >2 500 unique synthetic and natural-based scaffolds
• Specify MW limits of screening compounds
• Define any other inclusion/exclusion parameters
• Be supplied with up to 200 – 500 compounds per scaffold
• Choose to have compounds supplied exclusively or non-exclusively
• Define timeframe of the project

With a network of nearly 3,000 chemists in well-equipped labs across East Europe and Central Asia states, Interbioscreen has the resources, expertise and infrastructure to synthesize screening compounds from the scaffolds of your choice. Scaffold approach is not combinatorial chemistry. This is a custom synthesis project, which means that if you select any number of scaffolds from the database, the chemist will synthesize from 100- 500 derivatives of that structure. Syntheses can be very different, some involving to 12 steps. The most value of this project is in that the synthesis is performed in accordance with the customer’s guidelines /filters. Each company has its own objectives and thus specific requirements to the compounds they would wish to obtain. Since the selection of scaffolds is customer’s, this means that the customer is the first to receive custom-made compounds of their choice.

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Scaffolds Library Collections

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Building Blocks

Building Blocks

The Building Blocks (BB) collection currently contains
over 13200+ natural and synthetic in-stock-available building blocks.

The classification of the Building Blocks is indicated in the following fields.

  • PA  Primary Amines
  • SA  Secondary Amines
  • AL  Alcohols
  • AK  Aldehydes & Ketones
  • CA  Carboxylic Acids
  • ES  Esters
  • AN  Anhydrides & Haloanhydrides
  • PH  Phenols
  • AA  Aminoacids & their derivatives
  • PE  Peptides & their derivatives
  • CH  Carbohydrates & their derivatives
  • NU  Nucleotides & Nucleosides
  • ST  Steroids & Terpenoids
  • HAE  Haloid Alkyles & Epoxydes
  • SH  Thiols
  • OCC  Other Chemical Classes or Polyfunctional reagents
  • HY  Hydrazines & Hydrazones
  • IC  Isocyanates
  • ITC  Isothiocyanates
  • SC  Sulfochlorides

Search the Building Blocks collection using Chemical Formula or Catalog Number (Please wait while the pdf document loads)

Building Blocks Collections
(You can download the SDF file by scrolling to the bottom.)

Building Blocks Search:  Enter a list of catalog numbers, InChI, InChIKey or SMILES

Bioactive Compounds

Bioactive Compounds

The Bioactive Compound (BIO) collection includes known potent enzyme substrates, inhibitors and activators, receptor agonists and antagonists, bioregulators and other biologically active compounds for use in assay development and validation.

BIO database currently contains 789 compounds. In addition to the chemical and physical properties, each compound is accompanied by data on its biological use, mechanism of action, biological source (where applicable) and literature references.

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Bioactive Compounds  Collections

Bioactive Compounds Search:  Enter a list of catalog numbers, InChI, InChIKey or SMILES

Natural Compounds

Natural Compounds

The Natural Compound (NC) collection is one of the world’s largest and this fact has been acknowledged by the companies which pursue screening programs in Japan, Europe and the US.

IBS started to build up the NC collection in 1984, and to-date it has grown to include over 68000 compounds. Originally the main contributors of natural compounds & derivatives were research institutes of the former Soviet Union, e.g. institutes for plant chemistry, bioorganic chemistry, botanical institutes, institutes for biochemistry of microorganisms, phytopathology of plants etc. With time, we have expanded our sources of rare and unique natural compounds and over the past decade the collection has considerably grown due to the influx of natural products from Asia, Latin America, some European centers for natural products etc. In percentage terms, the collection can be said to comprise 30 – 35% of strictly natural compounds isolated from plants, microorganisms, marine species etc.; approximately 40% are derivatives of natural compounds, i.e. modified alkaloids, terpenoids, flavonoids etc, and the remaining 25-30% are mimetics (analogs) of the strictly natural compounds, e.g. azosteroids, azocoumarins, conjugated isoindole systems, oxaterpenoids etc.

Of the whole NC library, 60 – 65% are compounds of plant origin; 5-10% were isolated from microorganisms, about 5% from marine species and the rest from other natural sources. All compounds have purity at 92-98%. The structures and stereochemistry are confirmed by various physicochemical analytical methods, including NMR (Brucker 300 – 500 MHz), mass spectroscopy and in some cases X-ray analysis.

The most representative classes include various types of alkaloids (several thousand compounds), terpenoids (several thousand), flavonoids and coumarins (over 3 000 compounds), peptides, glycosides and nucleosides (over 1 000 compounds), phenol compounds (several hundred). The collection is also rich in rare and unusual compounds, such as various classes of phytoalexins, allelopathic agents, specific sex attractants, natural toxins, unusual sesquiterpenoids and other secondary metabolites. Apart from this, we also have a collection of functionalized natural compounds which can be used in Natural Combinatorial Chemistry as natural matrices. Compounds in this collection are available in the amounts from several grams up to several hundred grams. As a rule, we package natural compounds in vials or microplates as 2 mg – 5 mg – 10 mg – 20 mg – 50 mg samples.

 

Natural Compounds Collections

Natural Compounds  Search:  Enter a list of catalog numbers, InChI, InChIKey or SMILES

Synthetic Compounds

Synthetic Compounds

The Synthetic Compound (SC) collection contains over 485000 immediately available compounds.

Part of the integrated IBS SC collection is composed of the best of the Syntest Ltd., NELBI Ltd., ChemEx Inc., ExiMed Ltd. collections. Interbioscreen has purchased these selected sub-collections which are now Interbioscreen’s property. The synthetic library of IBS is built around functionalized heterocyclic drug-like molecules. Most of these cannot be obtained combinatorially. From the very start, IBS has been generating its library by making a very strict selection of the most interesting classes of compounds which have potential of becoming new drugs or plant protection agents or preparations for use in veterinary. We were less interested in simple hydrazones, hydrazides, Schiff’s bases or readily decomposing compounds. The major criterion for the inclusion of a compound in the collection has been the novelty, abundance in pharmacophores, diversity and the “drug-like molecule” structure.

Synthetic Compound (SC) collection

Synthetic Compound (SC) Search:  Enter a list of catalog numbers, InChI, InChIKey or SMILES

Compounds

IBS Interbioscreen Logo

World leading provider of high quality chemical libraries for screening programs since 1998

SYNTHETIC COMPOUNDS

The Synthetic Compound (SC) collection contains over 485000 immediately available compounds.

NATURAL COMPOUNDS

The Natural Compound (NC) collection is one of the world’s largest and this fact has been acknowledged by the companies which pursue screening programs in India, Japan, Europe and the US.

BUILDING BLOCKS

The Building Blocks collection currently contains over 13000 natural and synthetic in-stock-available building blocks.

BIOACTIVE COMPOUNDS

The Bioactive Compound (BIO) collection includes known potent enzyme substrates, inhibitors and activators, receptor agonists and antagonists, bioregulators and other biologically active compounds for use in assay development and validation.

Scaffolds Library

To meet customers’ drug discovery needs better, InterBioScreen Ltd. offers you the opportunity to obtain compounds synthesized around the Scaffolds from InterBioScreen’s libraries.

A Better Way to Isolate DNA From Plant Tissues

CTAB PROTOCOL FOR ISOLATING DNA FROM PLANT TISSUES

THE CONVENTIONAL PLANT DNA EXTRACTION METHOD AND A STREAMLINED ALTERNATIVE

Modern genomics techniques have promised to revolutionize plant biology, generating data to accelerate crop improvement, optimize plant selection, and advance our basic understanding of plant biology.1 Such techniques and applications rely on the extraction of high-quality DNA from a variety of distinct plant species and sample types.

To keep up with this rapidly advancing field, DNA extraction protocols must be robust, flexible, consistent, and fast. But plant tissues pose several challenges for even the most tried-and-true DNA extraction protocols. Plant cell walls are very difficult to break down and the cells contain many compounds that impede extraction and inhibit downstream molecular biology applications.

The CTAB Method: DNA Extraction from Plant Leaves and Seeds

To overcome the challenges presented by plant tissues, the cetyltrimethylammonium bromide (CTAB) method has become the “go-to” protocol for DNA extraction and purification from leaves and seeds. It was developed in the 1980s and has been used ever since, with various modifications for different plant species.2-5

In the CTAB procedure, the first step is breaking down the tissue, and it involves freezing your plant sample using liquid nitrogen. Once the tissue has been frozen, it’s ground into a fine powder with a mortar and pestle or a blender. After grinding, the tissue is transferred to a tube and CTAB buffer is added. The CTAB buffer facilitates cell lysis and prevents secondary metabolites from interfering with DNA extraction and downstream procedures.

Following plant cell lysis, RNase A is added to digest RNA, and DNA is separated from other cellular components using phenol/chloroform extraction, which separates the sample into two distinct aqueous and organic phases after centrifugation. Nonpolar molecules migrate into the organic phase and leave behind DNA and other polar molecules in the aqueous phase. The extraction is repeated on the aqueous phase until it becomes completely clear, and all DNA is collected. The aqueous phase is collected, and the recovered DNA is precipitated out with isopropanol. The precipitate is pelleted by centrifugation and washed with 70% ethanol to remove salts introduced during extraction. Once the ethanol is decanted, the residual ethanol in the pellet is evaporated away and the dried pellet is resuspended in your buffer-of-choice for your downstream application, such as PCR or NGS.

Considerations Before Performing CTAB DNA Extraction

The CTAB method is biochemically simple, easy to learn, and relatively cheap to perform. However, in practice, the protocol has several drawbacks: it’s lengthy, tedious, and low throughput, with many steps that require careful handling, exposure to hazardous chemicals, and several other technical considerations.

Timing Your DNA Extractions

From grinding with a mortar and pestle to resuspending sticky DNA pellets, the full CTAB protocol can take approximately two hours to process a small number of samples. There are also more than 20 steps in the protocol and as the number of samples increases, the amount of time needed to complete DNA extraction increases substantially. Plan your day carefully and set aside the proper amount of time to complete the entire protocol.

Working With Hazardous Materials

Caution must be taken when working with liquid nitrogen for the first grinding step as it can rapidly freeze skin tissue and cause cold burns even with short exposure. In addition, working with phenol and chloroform is also a biosafety hazard: Phenol can cause chemical burns and chloroform is a potential carcinogen.6,7 For many food testing labs, the use of these toxic chemicals is a major concern. Use of phenol/chloroform also generates organic waste which requires special storage containers and disposal procedures. Be sure you have the proper safety protocols in place before starting your DNA extractions.

Processing the Plant Tissue

Tissue grinding can vary between samples, leading to significant variation in extraction efficiencies and quality of DNA. To achieve more consistent tissue disruption across samples, you can also use a blender, though this step is still low throughput and time consuming. The more finely your tissue is ground, the more efficient you DNA extraction will be, making this a critical step for successful DNA extraction.

Phase Separation

A solution of phenol/chloroform/isoamyl alcohol is used to extract plant DNA from cellular debris and once added and vortexed, the mixture separates into three distinct phases: aqueous, interphase, and organic phase. While removing the aqueous phase and repeating the extraction is time consuming and laborious, it can also be challenging to remove all the aqueous phase, without disturbing the interphase. As a result, you may leave DNA behind or carryover contaminants from the interphase and organic phase, lowering your overall DNA yield and quality.

PCR Inhibitors

Several classes of biochemicals from plant tissues – polysaccharides, lipids, polyphenols, and/or other secondary metabolites – can coprecipitate with DNA, which can inhibit downstream applications that rely on thermostable DNA polymerases, such as PCR. The structure and concentration of these compounds can also vary substantially between different plant species, making the development and optimization of a “one size fits all” CTAB protocol difficult.8,9 In addition, phenol and other salts introduced throughout your CTAB protocol can remain, even after extensive ethanol washes. These impurities can also interfere with downstream applications, including PCR and NGS.10

Zymo’s Alternative Method for Isolation of DNA From Plant Tissues

If that all seems like a bit much, you aren’t wrong. Happily, there are plant-specific DNA isolation kits which provide faster, more consistent, high-purity DNA extraction than the conventional CTAB protocols and variations thereof. These are essential for maintaining and further supporting the rapidly evolving pace, scope, and scale of agricultural R&D.

Zymo’s Quick-DNA Plant/Seed kits use bead beating and column-based purification to provide a simple, rapid workflow for the isolation of inhibitor-free DNA from a variety of plant sources (Figure 1). There are no repetitive and lengthy phase separation steps or hazardous reagents used, so you can further streamline your lab’s operations and protect the safety of key personnel. You can also skip the lengthy RNase digestion, incubation and centrifugation periods, and precipitation steps.

Zymo’s Alternative Method for Isolation of DNA From Plant Tissues If that all seems like a bit much, you aren’t wrong. Happily, there are plant-specific DNA isolation kits which provide faster, more consistent, high-purity DNA extraction than the conventional CTAB protocols and variations thereof. These are essential for maintaining and further supporting the rapidly evolving pace, scope, and scale of agricultural R&D. Zymo’s Quick-DNA Plant/Seed kits use bead beating and column-based purification to provide a simple, rapid workflow for the isolation of inhibitor-free DNA from a variety of plant sources (Figure 1). There are no repetitive and lengthy phase separation steps or hazardous reagents used, so you can further streamline your lab’s operations and protect the safety of key personnel. You can also skip the lengthy RNase digestion, incubation and centrifugation periods, and precipitation steps.

Figure 1. Our simple plant and seed DNA purification workflow is great for diverse sample types and delivers high-quality DNA for several downstream applications.

The DNA extraction protocol can be completed in as little as 15 minutes and will give you a straightforward path to high-quality DNA. If you’re working with a challenging plant species, such as cacao and cannabis, try out our state-of-the-art BashingBeads for more complete lysis and more consistent yields (Figure 2).11-14

Figure 2. The Quick-DNA Plant/Seed kit delivers high-performance DNA from the most challenging plant species.

Our binding chemistry, wash solutions, and spin column technology remove polysaccharides, lipids, and other common downstream inhibitors and contaminants providing ultra-pure DNA, with minimal loss. Furthermore, our protocol has been optimized to work with a wide range of plant species and sample types, enabling novel and rapid advancements in modern plant genomics. See how we can help you extract high-quality plant DNA, in less time.

LEARN MORE ABOUT ZYMO’S PLANT DNA ISOLATION KITS HERE:

REFERENCES

 

  1. Genomics Era for Plants and Crop Species – Advances Made and Needed Tasks Ahead. IntechOpen website: https://www.intechopen.com/chapters/49877. Published July 14th, 2016. Accessed October 27th, 2021.
  2. Doyle J, Doyle J. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochem Bull. 1987;19(1):11-15.
  3. Murray MG and Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic acids research. 1980; 8(19); 4321–4325. https://doi.org/10.1093/nar/8.19.4321
  4. Aboul-Maaty NAF, Oraby HAS. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull Natl Res Cent. 2019;43(25). doi.org/10.1186/s42269-019-0066-1
  5. Muhammad I, Zhang T, Wang Y, et al. Modification of CTAB protocol for maize. Res J Biotech. 2013;8:41–45.
  6. 22. Safe Use of Phenol | Safety Services. UC – Davis Safety Services website: https://safetyservices.ucdavis.edu/safetynet/safe-use-of-phenol. Published March 26th, 2020. Accessed October 27th, 2021.
  7. Report on Carcinogens, Fourteenth Edition. National Toxicology Program website: https://ntp.niehs.nih.gov/ntp/roc/content/profiles/chloroform.pdf. Published November 3rd, 2016. Accessed October 27, 2021.
  8. Aboul-Maaty NAF, Oraby HAS. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull Natl Res Cent. 2019;43(25). doi.org/10.1186/s42269-019-0066-1
  9. CTAB Protocol for the Isolation of DNA from Plant Tissues. OPS Diagnostics website: https://opsdiagnostics.com/notes/protocols/ctab_protocol_for_plants.htm. Accessed October 27, 2021.
  10. Angeles JGC, Laurena AC, Tecson-Mendoza EM. Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.). Plant Mol Biol Report. 2012;23(3):297-298. doi:10.1007/BF02772760
  11. Kamber T, Malpica-López N, Messmer MM, et al. A qPCR Assay for the Fast Detection and Quantification of Colletotrichum lupini. Plants. 2021;10(8):1548. doi:10.3390/PLANTS10081548
  12. Romero Navarro JA, Phillips-Mora W, Arciniegas-Leal A, et al. Application of Genome Wide Association and Genomic Prediction for Improvement of Cacao Productivity and Resistance to Black and Frosty Pod Diseases. Front Plant Sci. 2017;8:1905. doi:10.3389/fpls.2017.01905
  13. Cornejo OE, Yee MC, Dominguez V, et al. Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process. Commun Biol. 2018;1:167. doi:10.1038/s42003-018-0168-6
  14. Garfinkel AR, Otten M, Crawford S. SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L. Genes. 2021;12(2):228. doi:10.3390/GENES12020228