dna extraction by ctab method ppt
Muhammad I, Zhang T, Wang Y, et al. Chloroform: Mixture of chloroform and isoamyl alcohol (24:1 ratio) equilibrated with a layer of TE buffer (pH 8) on top. The aqueous phase is collected, and the recovered DNA is precipitated out with isopropanol. Corpus ID: 102206113; Comparison of Four Methods of DNA Extraction from Needles of Pinus tabulaeformis @article{Ming2012ComparisonOF, title={Comparison of Four Methods of DNA Extraction from Needles of Pinus tabulaeformis}, author={Li Ming}, journal={Hubei Agricultural Sciences}, year={2012} } About Press Copyright Contact us Creators Advertise Developers Terms Privacy Policy & Safety How YouTube works Test new features NFL Sunday Ticket Press Copyright . See how we can help you extract high-quality plant DNA, in less time. [*ZeJ3"P|7He ]nBx)rgrRSfh3sVPkYQxs811Bwiw"gML xMCs34BA^Co{Ba[*- 8`EAr%EVCq)5U J)\)`.7 Activate your 30 day free trialto continue reading. Lysis of cell membrane: The first step of the DNA extraction is the rupture of the cell CTAB serves as an important surfactant in the DNA extraction buffer system to remove membrane lipids and promote cell lysis. Clipping is a handy way to collect important slides you want to go back to later. x0pnzmM )3jo.o8e'nn/j!;+RRRJ8F9k^ iN Qfe=\%A9BL YLoa 8C c)o63Z1`*4#q U0 For the extraction and purification of DNA from leaves and seeds, cetyltrimethylammonium bromide (CTAB) is the "go-to" procedure. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Treat the sample with PCI, centrifuge it, and collect the supernatant part. 6R(D$2(>1bD44b9!nGH-NCgVw IIOCebfcDKpC]rY8& "7 k@- We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. Transfer the supernatant to a new eppendorf tube and add 600 L chloroform, mix and spin at After it becomes homogeneous, take the mixture into the 2ml Eppendorf tube. Tap here to review the details. 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. Magnesium is a cofactor for DNase. Mix vigorously and incubate in water bath at 65C for 1 hr. % The first, and now the most numerous, organisms isolated from deep-sea hydrothermal vents belong to the order Thermococcales, within the Euryarchaeota in the domain Archaea. The salting-out method is a non-toxic DNA extraction method described by Miller, Dykes, and Polesky in 1988. Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet. release of the genomic DNA. Under these Be careful, take the only supernatant, and dont mix the supernatant with the debris. The DNA-containing sample is added to 3mL of lysis buffer (0.4 M NaCl, 10 mM Tris-HCl pH 8.0 and 2 mM EDTA, pH 8.0), SDS and proteinase K. The mixture is incubated at 55-65 overnight. Spin at ~10,000 g in microfuge (cold if possible) for 10 min. Besides its role in DNA extraction, CTAB is used as an antiseptic (against bacteria and fungus), detergent and ingredient of cosmetics. Such studies stamp CTAB as a major constituent of plant DNA extraction. It is imperative that sufficient cells are collected. Sterile spatulas Nonpolar molecules migrate into the organic phase and leave behind DNA and other polar molecules in the aqueous phase. Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.). A proteinase K step could be added at the same point as the lysozyme step, or immediately after the lysozyme step (add prot. www.HelpWriting.net This service will write as best as they can. other metals binds magnesium. The DNA extraction methods used were Cetyltrimethylammonium bromide (CTAB) and Commercial Kit (Geneaid; Genomic DNA Mini Kit Tissue), with variations in the number of nematodes: 1, 5, 10, 15 and . JP`N @$;(O2@%/n&73PLK`K!:x &P1N{Yrp}r>\XpY3_,(\! method, providing an overview before delving deeper into the procedure in a step-by-step approach. Government Pharmacy College Sajong, Government of Sikkim, University of Horticultural Sciences, Bagalkot, Endosperm culture and somatic embryogenesis, Biotechnology experiments 2nd semester (LNMU Darbhanga), Genomic Dna Isolation From Blood, Bacteria and Plasmid DNA Isolation, Lectut btn-202-ppt-l16. In this paper, most frequently used techniques for DNA extraction from insect with different size and condition are outlined. In contrast to the original method, the . Tannin is actually the main culprit. ice-cold ethanol (1000 l) to precipitate DNA incubate at -20C for at least 1 hour. 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. A modified CTAB method was applied to carry out molecular marker analysis to isolate good-quality DNA from . Na-acetate: 3M sodium acetate in RO water, sterilise by autoclaving (dont need to adjust pH). abyssi strain GE5. Percentages are represented as w/v. But plant tissues pose several challenges for even the most tried-and-true DNA extraction protocols. You choose the most appropriate method ! 70C, 1 hour). Extraction of DNA from Infected Cassava and Banana from Cameroon . Wash cell pellet in TE (resuspend then pellet again) before lysis if nuclease activity is a problem. XeQ ucdavis. p|`ylk/?|+.NB/;3a"a~w Y"nX#%C %Ss`2*;2%R=aX&~-o%LZax [C? ?MlB`>8O`-51H>tr$Tt=eyP Le|\! Instead of fuzzy bands, clear and sharp bands can be achieved by using a pinch of CTAB into the SDS PAGE. 0000002963 00000 n EB wont give as much protection as TE, which also protects against nucleases. In addition, the efficiency of PVP increases greatly by adding beta-mercaptoethanol. The extracted algal DNAs obtained from SDS, CTAB, and DTAB methods were suitable for PCR amplification of 18S rDNA region. *n1cFPw << /Length 5 0 R /Filter /FlateDecode >> This step also removes alcohol-soluble salt. Enjoy access to millions of ebooks, audiobooks, magazines, and more from Scribd. Its better to leave a trace of ethanol and water in the pellet (and have it dissolve easily), than to remove all moisture, and then have great difficulty dissolving the pellet. 0000002471 00000 n The beta-mercaptoethanol remains in the supportive role, but effectively blocks the oxidation of tannin, thereby disallowing interaction with DNA. Thus even if your DNA is good and your PCR fails repeatedly, you would have to worry about tannin. The instructor can "cheerlead" to encourage ample cells are collected. CTAB method is an organic solvent-based extraction method that has health hazards and harmful properties. For example, cetyltrimethylammonium bromide (CTAB) and guanidium thiocyanate (GITC) are often included in protocols for DNA . DNA was isolated from leaves of 10 plant species ( Cuminum cyminum, Vigna aconitifolia, Pennisetum typhoides, Tecoma stans, Lycium barbarum, Anogeissus acuminata, Tecomella undulata, Zizyphus mauritiana, Phoenix dactylifera, and Eruca sativa) and a fungus ( Fusarium oxysporum) using the CTAB method. The successive treatment with 70% ethanol allows an Extraction: In this step, polysaccharides, phenolic compounds, proteins and other cell lysates In this case, after adding TE and NaCl to cell pellet, add lysozyme to 1 mg/ml, and incubate 1 hour at 37C. However, care must be taken while handling liquid nitrogen. Now add chilled isopropanol (70%), and a pinch of NaCl to the supernatant and precipitate the DNA by inverting the tube several times. And eventually helps plants to stand straight. Plenty of proven DNA extraction chemicals are available, you can choose according to your need. A General DNA extraction scheme includes cell lysis, removal of contaminant and DNA stabilizing, precipitation and elution steps. Dellaporta (1983) and Maroof et al., (1984) have optimized the existing protocols and developed excellent methods for plant DNA extraction. Despite the existence of a huge collection of Thermococcales strains, very few plasmidic elements are reported so far in the hyperthermophilic euryarchaeotes. 2. H'8R(|Zp{7OeCl _Y&!S5sUFF$>`! q;>:V^6FUW>!Aq2WF Murray MG and Thompson WF. Use proteinase K overnight at room temperature or for 2 hours at 60C. I will also explain variations and concentrations of different CTAB buffers that you can try and use. 2.8. Method of DNA extraction: Many different methods and technologies are available for the isolation of genomic DNA. ^ cQmG;Q5h7~S.KC10NdXwkx|%!UPvDPXN2_82q?Lz>O&=pq7^9vmA(PdLe m C1!j 3SK97vU|(TgEw'q>jh:k"a6==UDnMW+6i4~t5F&CfYn*OGB"GrL#~]mT$zUIa 6/iDbe h'*omlb_i*G'*MBP q08C q~x]ufAi=Y a3FanN(CSQY6YvQ*K=($b6(+R@6}IDBDD+MD]R@+?%|C,Hi C}dFf13WI ve^*G. 0000003202 00000 n Method: In the present study, CTAB protocol given by Doyle and Doyle (1990) was modified and used for the isolation of genomic DNA from five medicinal plants. We have used this procedure successfully with Pseudomonas stutzeri strains which did not give good DNA preps with SDS-based DNA extraction methods due to polysaccharide production. CTAB protocol for the isolation of DNA from difficult tissues (high levels of secondary metabolites or polysaccharides), herbarium and soil. There are two reasons for that. Add 1/10 volume Na-acetate (50 l) and 2 vol. Nederlnsk - Frysk (Visser W.), Applied Statistics and Probability for Engineers (Douglas C. Montgomery; George C. Runger), How to extract plant DNA using CTAB method, 7 Chap 11 (Bioenergetic) F.Sc 1st Year Biology Helping Notes, L.N.Gumilyov Eurasian National University, Kwame Nkrumah University of Science and Technology, Jomo Kenyatta University of Agriculture and Technology, Constitutional law of Ghana and its history (FLAW306), Probability and statistics for engineers (MKT3802), Business Communication & Interpersonal Skills (MCM 509), Avar Kamps,Makine Mhendislii (46000), Power distribution and utilization (EE-312), Chapter 03 - The Time Value of Money (Part 1), SMA 2231 Probability and Statistics III course outline. A micro-level optimization is required to use it for our use. By whitelisting SlideShare on your ad-blocker, you are supporting our community of content creators. Start with sample preparation, clean the tissue and prepare it for homogenization. Along with CTAB, chemicals like SDS and PVP gave an excellent yield for Corn and Soybeans. Sterile pestle and mortar Plant cell walls are very difficult to break down and the cells contain many compounds that impede extraction and inhibit downstream molecular biology applications. Lysis: Tissue grinder and use detergent. pH 8.0 Autoclaved. This method will help the researchers to efficiently isolate DNA from . Liquid nitrogen can burn the skill and even do permanent damage. The solid phase protocol listed below is an alternative. You can also skip the lengthy RNase digestion, incubation and centrifugation periods, and precipitation steps. Saturated NaCl was needed for the proteins to precipitate out of the solution. But differences in the cell structure and chemical composition of a cell require a different DNA extraction setup. PRESENTED BY: AYESHA KABEER Increasing the heat and duration of the CTAB step might help with tough cells (eg. (CTAB) contained in the extraction buffer. 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. DNA extraction by chromatography: 2. [2] CTAB binds to the polysaccharides when the salt concentration is high, thus removing polysaccharides from solution. By whitelisting SlideShare on your ad-blocker, you are supporting our community of content creators. Contrary, animal cells dont have turgor pressure, as it is not actually required. Other chemicals required in the process are SDS, protease enzyme, Phenol, chloroform, isoamyl alcohol and ethanol. Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however the fundamentals of DNA extraction remains the same. Free access to premium services like Tuneln, Mubi and more. So you do not need to waste the time on rewritings. It is a cationic detergent, and water and alcohol soluble. The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing, and put into a solution containing salt. DNA extraction. CTAB Method DNA extraction - Molecular Biology BCH- Isolation of plant genomic DNA by modified CTAB - Studocu How to extract plant DNA using CTAB method molecular biology isolation of plant genomic dna modified ctab method principle: the extraction of genomic dna from Skip to document Ask an Expert Sign inRegister Sign inRegister Home Basic steps for DNA extraction 3. They work in a similar fashion, apart from CTAB being a cationic detergent while SDS is anionic. endstream endobj 38 0 obj <> endobj 39 0 obj <>/Font<>/ProcSet[/PDF/Text]>>/Rotate 0/Type/Page>> endobj 40 0 obj <> endobj 41 0 obj <> endobj 42 0 obj <> endobj 43 0 obj <> endobj 44 0 obj <> endobj 45 0 obj <> endobj 46 0 obj <> endobj 47 0 obj <>stream addition of glycine and/or ampicillin to weaken cells. xref How To Extract DNA From Whole (Fresh), Frozen, Dried and Clotted Blood? Be sure you have the proper safety protocols in place before starting your DNA extractions. The method needs to be modified for use on Gram-positives or yeast etc, by adding on extra lysis treatments at the front end of the protocol. DNA extraction is a process of isolating DNA from any biological cell to use it for analysis like PCR, sequencing or restriction digestion. Afterward, the homogenized sample would be incubated with lysis buffer and protease enzyme for effective lysis of the cell wall. ORGANIC METHOD OF DNA EXTRACTION 2. It was created in the 1980s, and since then, several adjustments have been made for usage with . Click here to review the details. inactivation of cellular nucleases and separation of the desired genomic DNA from cellular Discard pellet and take supernatant in another tube, Wash the sample with the alcohol (minimum 2 wash). Also, prepare a 10% stock solution of PVP. CTAB Protocol for Isolating DNA From Plant Tissues, the cetyltrimethylammonium bromide (CTAB) method, various modifications for different plant species, development and optimization of a one size fits all CTAB protocol difficult. If DNA precipitates are not observed after the addition of the chilled alcohol, then place the tube at -20C for 15 to 20 minutes and re-precipitate it. Precipitation: In this final stage, the nucleic acid is precipitated from the solution. Use the manufacturers guide for use. The Fast ID Genomic DNA Extraction Kit is designed for whole grains, fruits, and vegetables, and the Fast ID Ultra DNA Extraction Kit (Catalog No. 1. | DNA Extraction Method 2. of 0 TE+ RNase (100 g/ml). Extraction of DNA, RNA, and protein is the basic method used in molecular biology. Add liquid nitrogen and grind vigorously and add a lysis buffer, simultaneously. The nucleic acid is separated and precipitated using alcohol followed by a few rounds of washing with 70% ethanol. A comparison of DNA extraction methods used in research labs as opposed to classroom labs. dissolved in the aqueous solution are separated from the lysis homogenate. CTAB Method Abbreviated: Cetyl trimethylammonium bromide Function: CTAB buffer is used to lyse plant cells in the same way SDS is used to lyse bacterial cells. DNA extraction is one of the first steps to many research, forensic or clinical analyses. Precipitation . UYZsOhq9_&`YWJ+lZ^m]QfPZ^&UTj`F,M^Mmh!.8MLAnJ^gM>LXI=uC2s Ck@1o(T|! Most of the time students fail to extract good DNA. Autoclave all the utilities prior to use including solutions. INSTRUCTION: Dr Tushar Chauhan is a Scientist, Scientific-writer and co-founder of Genetic Education. If you will be using it a lot, consider making multiple smaller aliquots, since repeated freeze/thawing will deteriorate the quality. Separation step (Extraction of Protein) Organic DNA Extraction: PCI. Other methods of DNA extraction include salting out , cesium chloride density gradients , and chelex 100 resin [16, 17]. CTAB based extraction buffers are widely used when purifying DNA from plant tissues. Chloroform is toxic by inhalation and will also damage eyes. SDS (sodium dodecyl sulfate) is an excellent anionic detergent that can lyse proteins. extraction method is a simple and non-toxic technique that isolates a high-quality DNA from the whole blood. Urea is yet another excellent lysis agent that can be used along and alone in DNA extraction. Modified DNA extraction protocol i. Preheat the 3 extraction buffer in water bath at 65 C. Add 400 l TE and 100 l NaCl (5 M) to pellet, resuspend cells by vortexing. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. It is made up of a long phenolic ring and secreted during tissue damage, thus during the lysis process. 2 0 obj We've updated our privacy policy. 22. Ethanol and NaCl are used to remove plants polysaccharides. 4 0 obj The more finely your tissue is ground, the more efficient you DNA extraction will be, making this a critical step for successful DNA extraction. The DNA was used in nested or single PCR assays to detect the 16S rRNA and SAP genes using the phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (Deng & Hiruki . Manufacturers recommend homogenization of samples with mortar and pestle in presence of liquid nitrogen. To make powder with liquid nitrogen or crush it in sterilized pestle mortar with 2 mL of oBrgc;ntf24t``D7Sw8C&NQn"1_* ?dNOHhc/s3 ^D{6n>`SFcMedqW%MT0P!X?K` FlThc0 'wU/n;@wP~0ypKO8gW>LKn:?~hlfTvU=a"or^FuP]I.u;H\"X3Mh]buUbWQ5,N,0P+A+SXEAZV:57{F:999(G!Eh#`Ln{abDH"{JKiBH;jA} }9\cY"1La 5- Washing . Various chemical constituents of DNA extraction buffer allow effective isolation. The precipitate is pelleted by centrifugation and washed with 70% ethanol to remove salts introduced during extraction. Report on Carcinogens, Fourteenth Edition. PowerPoint: Audio and Video Product Design: From Sketch to CAD . https://doi.org/10.1093/nar/8.19.4321. Centrifuge the sample at 25,000rpm for 5 to 8 minutes and transfer the supernatant into another tube. PowerPoint Essential Training (Office 365/Microsoft 365) Cell preparation and extraction techniques. step (Lysis of White blood cells, WBC) 3- Phase . Cell wall and nuclear membrane lysis- by chemical treatment, tissue homogenization using liquid nitrogen and physical grinding. Note also that all waste chloroform and phenol arising from this procedure must be discarded as hazardous chemical waste (not down the sink!). w;owwllND\| Ideal lysis procedure is rigorous enough to disrupt the complex starting material (plant 0000000812 00000 n Take 100 mg tissue, homogenate Safe Use of Phenol | Safety Services. Angeles JGC, Laurena AC, Tecson-Mendoza EM. _:)a"P$,4+ 5>15Kzv$& However, TE can interfere with subsequent enzyme reactions (EB wont). For teachers that have done DNA extraction from strawberry or thymus, fewer cells collected in this experiment. Notedly, keep in mind that the composition of the buffer and concentration of chemicals would differ from plant species to species. Take 5gms of fresh plant tissue and cut it into small pieces. Fast ID contains powerful denaturants and DNA-selective startxref A lysozyme incubation can be added if cells dont lyse well with CTAB alone. the DNA purity and therefore quality. After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE. Zymos 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). Add 500 l phenol:chloroform, vortex until milky solution obtained (~20 sec), spin at top speed in microfuge for 5 min, retain supernatant. Extraction(CTAB) Buffer 1.4 M Na Cl In this case, it is recommended to add an isopropanol precipitation step after the first chloroform extraction, then do a 70% ethanol rinse, dry pellet, redissolve in 1-2 ml TE. Water bath set at 65C Remember to boil RNAse before use to kill any DNAse in it (see Sambrook for method). 2- Digestion . 2. Mix gently to precipitate the nucleic acid. Lets see how it works and how to optimize the CTAB buffer for plant DNA extraction.. After the clear pellet appears, add 500L of TE buffer or elution buffer to the pellet and dissolve the DNA in it. ^ 0=pLRto6Q&N(Na*Y;"I$1k*wkX 5|AQ>4DWii/:&"E N Bl~oNiefef_ L>{$^bKb~dh~REBtz(4>/kP',2"32GnF(8xi9 Genomic DNA extraction and amplicon generation. Application of Genome Wide Association and Genomic Prediction for Improvement of Cacao Productivity and Resistance to Black and Frosty Pod Diseases. 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. Tap here to review the details. 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 . DNA extractions using a traditional CTAB-based method with (A) no PVP, (B) 1% PVP and (C) 4% PVP. The use of Urea is the easy and safest way to extract DNA from plants and sufficient enough for routine PCR and restriction digestion but not enough for sequencing or microarray analysis. The procedure for fixing the aptamer onto these transducers and for monitoring the interaction with the target protein is shown here. Plant cells can be lysed with the ionic detergent CTAB, which forms an insoluble complex with Now wash the DNA pellet with alcohol two times at 9000 rpm for 2 minutes. additional purification, or wash, of the nucleic acid from the remaining salt. We've encountered a problem, please try again. 5. Jun 2012 - Dec 2012. 0000000536 00000 n Select a plant species and prepare the protocol and chemicals accordingly. 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. Activate your 30 day free trialto unlock unlimited reading. We use this method for extracting genome sequencing quality (i.e. DNA Extraction - Cells are resuspended in 0.8 mL of pre-warmed (60C) CTAB extraction buffer CTAB buffer 2% CTAB (hexadecyltrimethylammonium bromide) 100 mM TrisHCl [pH=8] 20 mM EDTA, 0.2% -mercaptoethanol [added just before use] 1.4 M NaCl 0.1 mg/mL proteinase K [added just before use]) MilliQ or Nanopure) and filter sterilize (push solution Activate your 30 day free trialto unlock unlimited reading. Main Outcome Measures: Successful extraction of DNA Results: PCR, RFLP and DNA sequencing gave the expected results in all the DNA samples extracted by all the three methods (boiling, microwave irradiation and the commercial kit). Now customize the name of a clipboard to store your clips. Now let us come to our major player- CTAB. On with Nicks notes! Add 500 l chloroform, vortex, spin 5 min, retain supernatant. I already have explained the different types of polysaccharides and polyphenols present in plants and required chemical modifications accordingly. Separation is also successful when the tissue contains high amounts of polysaccharides. and transfer the powder or 600 L of For this purpose, the homogenized sample is first treated with the CTAB Protocol for the Isolation of DNA from Plant Tissues. Get news, product info, tips, industry updates, events, freebies, and more delivered right to your inbox. The structure is explained in the figure below. We've updated our privacy policy. So these three things (cell wall composition, secondary metabolites and turgor pressure) need advanced treatments. conditions, the detergent i. CTAB, which is more stable in alcohol than in water, can be washed (100 mM Tris-HCl pH 8, 2% (w/v) CTAB, 20 mM EDTA, 1 M NaCl, and 1% (v/v) hb```Lf| HITS-CLIP (CLIP-Seq) for mouse Piwi proteins. Stabilizing and precipitating DNA- by chemical treatment using NaCl and alcohol. separation of the aqueous and organic phases. In this study, we demonstrate a modified CTAB method for the isolation of DNA to maximize the quality and yield from small aphids.