dna extraction by ctab method ppt

dna extraction by ctab method ppt

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. 0000001976 00000 n Plant cell walls are very difficult to break down and the cells contain many compounds that impede extraction and inhibit downstream molecular biology applications. Only four small populations remain in Xue, Nanhu, and Cilai Mountains of Taiwan. DNA extraction is one of the first steps to many research, forensic or clinical analyses. After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE. It was developed in the 1980s and has been used ever since, with various modifications for different plant species.2-5. However, the CTAB-like buffer system can do better with such hard tissues. 1- Lysis . Free access to premium services like Tuneln, Mubi and more. This research evaluated five genome extraction methods including Protocol A (main CTAB), Protocol B (CTAB without -mercaptoethanol), Protocol C (CTAB without ammonium acetate), Protocol D (modified method of Murray and Thompson), and Protocol E (Gene All-Kit), to determine their ability to extract DNA from fresh and herbarium leaves of A. fridae. SDS (sodium dodecyl sulfate) is an excellent anionic detergent that can lyse proteins. Notedly, keep in mind that the composition of the buffer and concentration of chemicals would differ from plant species to species. (BBTV) using PCR RNA Extraction from infected Okra and Cabbage samples from Cameroon, using CTAB method of extraction Show more Show less Education Ladoke Akintola University of Technology . The hazard with traditional CTAB protocols is the protein component of plant lysates is usually removed using phenol and chloroform. ii. This plasmid, pGT5, is a stable multicopy plasmid (2530 copies per chromosome) and can be isolated from cultures at different growth phase and in the temperature range from 75 up to 105oC. For teachers that have done DNA extraction from strawberry or thymus, fewer cells collected in this experiment. Nonpolar molecules migrate into the organic phase and leave behind DNA and other polar molecules in the aqueous phase. 4. 0000002963 00000 n 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. Diversity and Selection of Shell of the Hermit Crab of Mandvi, Kachchh Coast, Doodle Astronomy Lesson XL by Slidesgo.pptx, A Pragmatic Approach for Solving the Sports Scheduling Problem-presentation.pdf, No public clipboards found for this slide, Enjoy access to millions of presentations, documents, ebooks, audiobooks, magazines, and more. CTAB buffer (preheated at 65C for 15 min.) IntechOpen website: https://www.intechopen.com/chapters/49877. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue . Copyright 2023 StudeerSnel B.V., Keizersgracht 424, 1016 GC Amsterdam, KVK: 56829787, BTW: NL852321363B01, Managerial Accounting (Ray Garrison; Eric Noreen; Peter C. Brewer), Principles of Marketing (Philip Kotler; Gary Armstrong; Valerie Trifts; Peggy H. Cunningham), The Importance of Being Earnest (Oscar Wilde), English (Robert Rueda; Tina Saldivar; Lynne Shapiro; Shane Templeton; Houghton Mifflin Company Staff), Mechanics of Materials (Russell C. Hibbeler; S. C. Fan), Auditing and Assurance Services: an Applied Approach (Iris Stuart), Marketing-Management: Mrkte, Marktinformationen und Marktbearbeit (Matthias Sander), Handboek Caribisch Staatsrecht (Arie Bernardus Rijn), Junqueira's Basic Histology (Anthony L. Mescher), Big Data, Data Mining, and Machine Learning (Jared Dean), Frysk Wurdboek: Hnwurdboek Fan'E Fryske Taal ; Mei Dryn Opnommen List Fan Fryske Plaknammen List Fan Fryske Gemeentenammen. Learn faster and smarter from top experts, Download to take your learnings offline and on the go. Edwards et al. For this purpose, the homogenized sample is first treated with the CTAB Protocol for the Isolation of DNA from Plant Tissues. CTAB protocol for the isolation of DNA from difficult tissues (high levels of secondary metabolites or polysaccharides), herbarium and soil. Tris-HCl gives the solution a pH Follow this protocol on how to use PCI: Phenol, chloroform and isoamyl alcohol DNA extraction. Garfinkel AR, Otten M, Crawford S. SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L. Doyle J, Doyle J. Academia.edu no longer supports Internet Explorer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. DNA quality was checked on 1% agarose gels using a ladder and the purity was checked as above. Precipitation . UNIVERSITY OF GUJRAT SIALKOT SUBCAMPUS. Alex: Gee, thats a lot of chloroform, might be a while before I risk trying this protocol. However, other homogenization such as rotor-stator homogenizer or bead mills can be suitable. https://doi.org/10.1093/nar/8.19.4321. 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. << /Length 5 0 R /Filter /FlateDecode >> Various chemical constituents of DNA extraction buffer allow effective isolation. Various chemical constituents of DNA extraction buffer allow effective isolation. But plant tissues pose several challenges for even the most tried-and-true DNA extraction protocols. %PDF-1.3 DNA extraction is a process of isolating DNA from any biological cell to use it for analysis like PCR, sequencing or restriction digestion. stream Institute of Biomedical and Genetic Engineering (IBGE), Dna fingerprinting matreilas & methods of chilli, Dna extraction from fresh or frozen tissues, Human biosafety issues of nanomaterials used as nanomedicine, Introduction, pathophysiology and treatment options for covid 19, Inhibition of Gene Expression at RNA Level. Step 1. Tap here to review the details. Let us see the protocol. How To Extract DNA From Whole (Fresh), Frozen, Dried and Clotted Blood? The SlideShare family just got bigger. Results from six grams of leaf tissue finely ground using a mortar and pestle, then aliquoted (1 g) for each extraction. Discard all waste phenol and chloroform into chemical waste bottle in fume hood. Method of DNA extraction: Many different methods and technologies are available for the isolation of genomic DNA. By centrifugation, such co-precipitates can be removed in consecutive steps.The structure of CTAB. This protocol is my own, we used it in our labs during our masters. c*f(M=;"]txGyFUHIEW8[WE%:8ioGSTzPF*G@ ioE DNA precipitates, resembling a thread of translucent white snot, at the interface between the juice and alcohol. of 0 TE+ RNase (100 g/ml). By whitelisting SlideShare on your ad-blocker, you are supporting our community of content creators. ?1tr XE.8TC!qC^t5W3C"w+| 4(XH@CaqB"'1"&)l7%$,Y$DNlNsRF =WPj'Y~u[.qA$D fd9AV'qSFD#>-JBpl W:LlC}P?.EBl!|J)GaABAUoR=$SXjg!K TQQcDxJ$Jh>64=6S[*z~0(OL The structure and concentration of these compounds can also vary substantially between different plant species, making the development and optimization of a one size fits all CTAB protocol difficult.8,9 In addition, phenol and other salts introduced throughout your CTAB protocol can remain, even after extensive ethanol washes. Enjoy access to millions of ebooks, audiobooks, magazines, and more from Scribd. Genomic DNA was extracted using the CTAB method (Doyle & Doyle, 1987), with some modifications, including a Sorbitol pre-treatment to remove mucilaginous polysaccharides (Souza & al., 2012). PROJECT ON WASTE WATER TREATMENT AND ENERGY PRODUCTION FROM SLUDGE AND SOLID FERMENTATION: TECHNIQUE USED FOR THE PRODUCTION OF FOOD BY MICROBES, Human glucose-6-phosphate isomerase(GPI) or phosphoglucose isomerase(PGI), PHOSPHOLIPIDS, GLYCEROPHOSPHOLIPIDS, SPHINGOMYELIN AND GLYCOLIPIDS, Simulations of Test Reduction Using Pooled Heavy Metals Analysis inCannabis, Quantized space time and Einstein's special theory of relativity.pdf. Use PCI (phenol: chloroform: isoamyl alcohol) in combination with the CTAB buffer. I will also explain variations and concentrations of different CTAB buffers that you can try and use. When the cell membrane is exposed to the 4UYG$(. inactivation of cellular nucleases and separation of the desired genomic DNA from cellular These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. Spin at top speed in microfuge (cold if possible) 10 min, drain off ethanol, retain pellet. Importantly, follow a common lab safely and perform the experiments using liquid nitrogen under the supervision of experts. EB (Elution buffer): 5 mM Tris-HCl. Other chemicals required in the process are SDS, protease enzyme, Phenol, chloroform, isoamyl alcohol and ethanol. Cell lysis is aided with a CTAB buffer, which also stops secondary metabolites from obstructing DNA extraction. EXTRACTION (i) Grind 2 to 5 g of frozen leaves to a very fine powder using N2 cooled mortar and pestle. Along with CTAB, chemicals like SDS and PVP gave an excellent yield for Corn and Soybeans. In this sense, several studies have reported the efficiency of using cocoa leaf material for DNA extraction, however, has not been considered leaf developmental stage as an important variable for obtaining high quantity and quality of DNA. So the first thing one should have to keep in mind is that depending upon the constituent of a plant cell wall, and the type of polyphenols they contain, prepare a buffer system. Instant access to millions of ebooks, audiobooks, magazines, podcasts and more. The procedure can also be easily scaled up tenfold to accommodate 50 ml broths. This paper is only a guide for these techniques and we describe. Phenol is volatile and can burn the skin. The precipitate is pelleted by centrifugation and washed with 70% ethanol to remove salts introduced during extraction. Clipping is a handy way to collect important slides you want to go back to later. Separation step (Extraction of Protein) Organic DNA Extraction: PCI. DNA extraction Method for plant sample using CTAB method. 48 0 obj <>stream 37 12 3) CTAB is widely used to disturb membranes and common method used in Plant DNA Extraction whereas SDS is used to solubilize membrane and used in Bacterial DNA Extraction, remove. The beta-mercaptoethanol remains in the supportive role, but effectively blocks the oxidation of tannin, thereby disallowing interaction with DNA. Looks like youve clipped this slide to already. The solid phase protocol listed below is an alternative. MATERIALS: INSTRUCTION: The more finely your tissue is ground, the more efficient you DNA extraction will be, making this a critical step for successful DNA extraction. Lysis: Tissue grinder and use detergent. 10 Different Types of DNA Extraction Methods (Updated), 7 Ways to Determine Genotypes Using Gel Electrophoresis, DNA Extraction From Dried Blood Spot Samples: Protocol + Comprehensive Guide, Metagenomics Made Easy: Streamlining DNA Extraction With Bead Beating, A Guide To Next-Generation Shotgun Sequencing In Metagenomics: Technique, Advantages and Challenges. tissue), yet gentle enough to preserve the target nucleic acid. 2023 Zymo Research Corporation. It was used to extract material for the Micromonas RCC299 complete genome sequencing project, and the Micromonas RCC472 genome sequencing project. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under eld Codon p referance and factor influences it, How to approach supervisors for research opportunities, Direct Lineage Reprogramming: Novel Factors involved in Lineage Reprogramming, A common molecular basis for three inherited kidney, Presentation insect resistant transgenic crops ahmad madni (21-12-2016), Field application of metal microbe interaction, PPT for COT Methods of Cooking Poultry.pptx, CUNY Brooklyn College Diagnostic Museum Report Between Two Paintings.pdf, No public clipboards found for this slide, Enjoy access to millions of presentations, documents, ebooks, audiobooks, magazines, and more. Plant cells can be lysed with the ionic detergent CTAB, which forms an insoluble complex with extraction method is a simple and non-toxic technique that isolates a high-quality DNA from the whole blood. Note: If the DNA does not clump together, students can draw up the solution with a pasteur pipette or an eye dropper. 2- Digestion . Do not sell or share my personal information, 1. Care must be taken while handling CTAB as it has corrosive and toxic properties. In the standard salting-out method, proteins K and RNase are added to them after the lysis of cells. addition of glycine and/or ampicillin to weaken cells. 0000000536 00000 n Add 500 l chloroform, vortex and mix thoroughly, incubate on ice 30 min. To achieve more consistent tissue disruption across samples, you can also use a blender, though this step is still low throughput and time consuming. [,' u8p;le n0%j7giL (0xS6mnf6tW choosing a selection results in a full page refresh. polysaccharides in a high-salt environment. the pellet In addition, plant DNA extraction is even a difficult task to execute because of their specialized cell structure and composition.

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