2003 Casita Spirit Deluxe $800, Riverside High School Teachers, Life Tenant Not Maintaining Property, Aries Sun, Virgo Moon Taurus Rising, Articles D

4. 6R(D$2(>1bD44b9!nGH-NCgVw IIOCebfcDKpC]rY8& "7 k@- Nonetheless, to get success every time in plant DNA extraction, the first thing is to understand the structure and constituent of the plant cell of the species you are using. Use proteinase K overnight at room temperature or for 2 hours at 60C. Gw.:S /vR.fP" pbgQ$C e;=AE-0I's>rEwd z.Ar1-tAMF.~V\2;MC#:- 54o)IR pZ|h!0"# Edwards et al. DNA was diluted to 1 ng l 1 with sterile distilled water. HITS-CLIP (CLIP-Seq) for mouse Piwi proteins. extraction and purification of DNA from plants and plant derived foodstuff and is particularly It was adapted from Winnepenninckx B. et al., 1993, TIG: 9 (12), 407 (Technical Tips). 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. See how we can help you extract high-quality plant DNA, in less time. Remember to boil RNAse before use to kill any DNAse in it (see Sambrook for method). Murray MG and Thompson WF. DNA quality was checked on 1% agarose gels using a ladder and the purity was checked as above. EB wont give as much protection as TE, which also protects against nucleases. overview before delving deeper into the procedure in a step-by-step approach. The DNA extraction protocol can be completed in as little as 15 minutes and will give you a straightforward path to high-quality DNA. mix gently by inverting and spin at 13,000 rpm at 4C for 10 min. J w9K|F>.-c#0G{3F)2jSV7\snE>rl gCe:"6zkO( It is made up of a long phenolic ring and secreted during tissue damage, thus during the lysis process. out, while the nucleic acid precipitates. 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. In addition, the presence of turgor pressure, all these things creates problems in plant DNA extraction. 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. Reagents 1. 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. For teachers that have done DNA extraction from strawberry or thymus, fewer cells collected in this experiment. There are two reasons for that. I already have explained the different types of polysaccharides and polyphenols present in plants and required chemical modifications accordingly. debris. QLnoSc3 TuAWbAJ7$ (5hiHFn%wCEJ}=PbcSB#hN(YYXl69Dr ]\79/SiSM J,QOY5#^A@r|)(' g@9_d[v9|L.Th*9I5D(Y Precipitation . JP`N @$;(O2@%/n&73PLK`K!:x &P1N{Yrp}r>\XpY3_,(\! (1991) used 0.5% SDS with the CTAB buffer and developed a simple plant DNA extraction protocol. K to 0.5 mg/ml, incubate 60C 1-16 h). DNA extraction Method for plant sample using CTAB method. Summary Aptamers are an alternative to antibodies in their role as biorecognition elements in analytical devices. if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[250,250],'geneticeducation_co_in-box-4','ezslot_21',149,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-box-4-0'); Go through this article: Why is DNA extraction from plants difficult? The proteinase K step is additional (you can use it if necessary). Click here to review the details. In principle, in the extraction process, CTAB associates with the cell wall polysaccharides and proteins and co-precipitates it along with DNA. Not sterilized. By centrifugation, such co-precipitates can be removed in consecutive steps.The structure of CTAB. Ideal lysis procedure is rigorous enough to disrupt the complex starting material (plant KIT METHOD OF DNA EXTRACTION. Remember that genomic DNA means all the DNA, so this method extracts both chromosomes and plasmids. And the soft membrane of the animal cells cant bear it. Discard all waste phenol and chloroform into chemical waste bottle in fume hood. Former website http://biogeek. However, too much ethanol is also BADDNA will float out of wells on gel. DNA extraction by chromatography: 2. The basic principle of DNA isolation is disruption of the cell wall, cell membrane, and nuclear membrane to release the highly intact DNA into solution followed by precipitation of DNA and removal of the contaminating biomolecules such as the proteins, polysaccharides, lipids, phenols, and other secondary metabolites by enzymatic or chemical In addition, the efficiency of PVP increases greatly by adding beta-mercaptoethanol. Same volume of isopropanol is added into supernatant to make DNA pellet. Leaves were disrupted using Qiagen TissueLyser II. It appears that you have an ad-blocker running. step (Lysis of White blood cells, WBC) 3- Phase . In the CTAB procedure, the first step is breaking down the tissue, and it involves freezing your plant sample using liquid nitrogen. Report on Carcinogens, Fourteenth Edition. Saturated NaCl was needed for the proteins to precipitate out of the solution. suitable for the elimination of polysaccharides and polyphenolic compounds otherwise affecting In this study, we compared three different protocols of DNA extraction: NaCl (saline); phenol-chloroform and commercial kit (Promega)from three biological tissues of five individuals of Lutjanus purpureus under two methods of storage. You choose the most appropriate method ! Pre-prepare a CTAB solution (10%) by heating and store it at 37C temperature. 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 . Proteinase K is yet another excellent ingredient that also can digest proteins- DNase and RNase present in the cell and protects the nucleic acid from degradation. The beta-mercaptoethanol remains in the supportive role, but effectively blocks the oxidation of tannin, thereby disallowing interaction with DNA. Salting-out method. 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 Download our ebook: From DNA Extraction to PCR, Plant DNA extraction needs a CTAB DNA extraction buffer that contains ingredients that can effectively remove polysaccharides and polyphenols. JuMRUN.)oMf91 E@4E+t5mAc5fJHI3?K#Sk[~Php#3D t jhPd|c|4bHVOQE%M%Zh**W0o-B 0ZN&1 w;owwllND\| Nonpolar molecules migrate into the organic phase and leave behind DNA and other polar molecules in the aqueous phase. the DNA purity and therefore quality. 1980; 8(19); 43214325. By whitelisting SlideShare on your ad-blocker, you are supporting our community of content creators. We've encountered a problem, please try again. 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. iKjA3)])"ZcaR*.&p)n5x.9,Vjt_* K;lZe0# ;Iv1WUbg>s{x,4L4(@[xidmy)dK4^Cv$[1` 8[O16Gok9$pZBQM'!(H0RK9ra)"}Vjo7`/J1j#3g3]( eo; M(]+d[` mAQ8 rk-%wURl0]vcRBqR^W(1+.Ho)T-^gbrGvVp-{x%`1wE;b,uD^Y{_o 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. Let me know if it works should you try it though! This paper is only a guide for these techniques and we describe. Tissue grinding can vary between samples, leading to significant variation in extraction efficiencies and quality of DNA. Ch05 - Chapter 05 solution for Intermediate Accounting by Donald E. Kieso, Jerry J. About Press Copyright Contact us Creators Advertise Developers Terms Privacy Policy & Safety How YouTube works Test new features NFL Sunday Ticket Press Copyright . They work in a similar fashion, apart from CTAB being a cationic detergent while SDS is anionic. Note also that all waste chloroform and phenol arising from this procedure must be discarded as hazardous chemical waste (not down the sink!). Kamber T, Malpica-Lpez N, Messmer MM, et al. Be careful, take the only supernatant, and dont mix the supernatant with the debris. By accepting, you agree to the updated privacy policy. We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. 3. dissolved in the aqueous solution are separated from the lysis homogenate. Nguyen Quoc Trung. 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. In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less These two solvents are generally considered hazardous. Protocol for Genomic DNA extraction with CTAB NeoSynBio This protocol can be used on most gram-negative bacteria to obtain high-quality genomic DNA, which is both pure and very high molecular weight. Prepare a 10X stock for CTAB solution to avoid repeated chemical preparation. Previous studies demonstrated that Tannin is a proven PCR inhibitor that suppresses PCR amplification and remains bound with the DNA. Increasing the heat and duration of the CTAB step might help with tough cells (eg. However, TE can interfere with subsequent enzyme reactions (EB wont). DNA extractions using the NGS protocol with (D) no PVP, (E) 1% PVP, and (F) 4% PVP. Add the beta-mercaptoethanol just before the use. 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. Phenol is toxic and corrosive to skin and eyes. Lysis: Tissue grinder and use detergent. Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.). Liquid nitrogen can burn the skill and even do permanent damage. And eventually helps plants to stand straight. separation of the aqueous and organic phases. These are essential for maintaining and further supporting the rapidly evolving pace, scope, and scale of agricultural R&D. It was created in the 1980s, and since then, several adjustments have been made for usage with . Overdrying of the final DNA pellet is BAD. 2. I .g$!=n]BQ|nTr|0T5t4@XIR$fwyVIszC9Er(ze*RzoI$M%8FM=Mi fR{X[:[F R=XFBdvk>*H7&NIP'B.4GsXUS6j'^p2+#hY[xBYUJuk (L)4/vB`])tS2\YI!MSCKVN",NAN}/DKHFR Method of DNA extraction: Many different methods and technologies are available for the isolation of genomic DNA. Extraction(CTAB) Buffer 1.4 M Na Cl Techniques covered include genomic DNA extraction using cetyl trimethylammonium bromide (CTAB) and chloroform extraction, chromatographic techniques, ELISA, hybridization, gel electrophoresis, dot blot analysis and methods for studying polymerase . For DNA, absorbance at A 260 (also called optical density, OD) is converted into DNA concentration by following method: A 260/OD of 1.0 = a concentration of 50 g/ml of double-stranded DNA . A total of 30 samples of five algal genus were used in this study. It was developed in the 1980s and has been used ever since, with various modifications for different plant species.2-5. 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. CTAB serves as an important surfactant in the DNA extraction buffer system to remove membrane lipids and promote cell lysis. Wash cell pellet in TE (resuspend then pellet again) before lysis if nuclease activity is a problem. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. The following stock solutions and reagents were prepared by mixing the standard amount of chemicals. Precipitating the DNA with an alcohol usually ice-cold ethanol or isopropanol. On with Nicks notes! The method is fast,. Genomic DNA extraction and amplicon generation. UYZsOhq9_&`YWJ+lZ^m]QfPZ^&UTj`F,M^Mmh!.8MLAnJ^gM>LXI=uC2s Ck@1o(T|! 22. 5. DNA must be purified from cellular material in a manner that prevents degradation. -mercaptoethanol). Instant access to millions of ebooks, audiobooks, magazines, podcasts and more. NaCl. In contrast to the original method, the . Add 0.3% 2--mercaptoethanol to the 3 CTAB extraction buffer immediately before use. )Z$m|h_8"dOtv#P%w;:K&z]x.A`5.AZ%q9;eU! Decant and dry the pellet at RT. Take 100 mg tissue, homogenate Once the nucleic acid complex has been CTAB buffer (preheated at 65C for 15 min.) Clipping is a handy way to collect important slides you want to go back to later. Prepare a working solution from stock. Molecular genetic techniques are an effective monitoring tool, but high-quality DNA samples are usually required. IntechOpen website: https://www.intechopen.com/chapters/49877. METHODS OF DNA EXTRACTION Free access to premium services like Tuneln, Mubi and more. DNA extraction is a process of isolating DNA from any biological cell to use it for analysis like PCR, sequencing or restriction digestion. insoluble complex with the polysaccharides. DNA CHECK RUN: 11. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA. Now customize the name of a clipboard to store your clips. (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 . Dr Tushar Chauhan is a Scientist, Scientific-writer and co-founder of Genetic Education. 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. Other methods of DNA extraction include salting out , cesium chloride density gradients , and chelex 100 resin [16, 17]. https://doi.org/10.1093/nar/8.19.4321. Plate-grown cells are fine also growth scraped from half of a streak-plate is about the same amount of biomass as a fully-grown 5 ml broth. You can read the details below. Angeles JGC, Laurena AC, Tecson-Mendoza EM. %%Jb4jgAgluFd&Hw4[hply_.FnZL1ywbIWuB}^zl|pq. 0000002471 00000 n Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Plant samples (leaf, callus etc.) 0000001496 00000 n The salting-out method is a non-toxic DNA extraction method described by Miller, Dykes, and Polesky in 1988. I hope to test this at some point to confirm some of these comments, but as my source for many of these protocols, I know he is rarely, if ever wrong. Magnesium is a cofactor for DNase. Results from six grams of leaf tissue finely ground using a mortar and pestle, then aliquoted (1 g) for each extraction. Clipping is a handy way to collect important slides you want to go back to later. Centrifuge the precipitate for 10,000rpm for 2 minutes and collect the pellet (remove the supernatant). The mantra to success in plant DNA extraction is to grind tissue well, hard, and rough until fine homogenization. What is the Role of Alcohol in DNA extraction? This will increase the yield. edu/Mstats/. xref Enjoy access to millions of ebooks, audiobooks, magazines, and more from Scribd. Wash with around 700 L of 70% ethanol. Incubate 1 hr at 37C. Techniques of DNA Extraction, Purification and Quantification, Nucleic Acid Quantification Methods - DNA / RNA Quantification, Isolation and purification of microbial c, DNA- Basics on isolation, quantification, storage, Application of molecular technology in biotechnology, Sarhad University of Science and Technology, Biotechnology experiments 2nd semester (LNMU Darbhanga), International Medicine School - Management and Science University, Dna extraction strawberry lab spring 2015, Extraction buffer, Protease inhibitors methods of cell distrubtion, KYBELLA AND OTHER FILLERS USED FOR BEAUTY ENHANCEMENT. A fragment analyzer Qsep100, that uses a capillary gel electrophoresis system, allowed . Why is DNA extraction from plants difficult? It appears that you have an ad-blocker running. May need to be heated to 50C to dissolve. ?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 Chloroform: Mixture of chloroform and isoamyl alcohol (24:1 ratio) equilibrated with a layer of TE buffer (pH 8) on top. 0000003202 00000 n Breaking cells open to release the DNA. All Rights Reserved. Phenol, chloroform and isoamyl alcohol DNA extraction. a vGX. To browse Academia.edu and the wider internet faster and more securely, please take a few seconds toupgrade your browser. The aqueous phase is collected, and the recovered DNA is precipitated out with isopropanol. 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. EXTRACTION (i) Grind 2 to 5 g of frozen leaves to a very fine powder using N2 cooled mortar and pestle. compounds and other contaminants remain in the supernatant and can be washed away. Stabilizing and precipitating DNA- by chemical treatment using NaCl and alcohol. (Modification of "CTAB method", in Current Protocols in Molecular Biology) Cell growth: To minimize gDNA sampling bias (e.g., excess coverage of sequences around the origin of replication) please take precautions NOT to proceed with DNA isolation while most of the cell population is in the ), and the procedures can be done in microfuge rather than big centrifuge (faster!). 2 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 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. You have to modify the protocol and preparation as per your need. For this Q TP? CTAB extraction buffer. As a result, you may leave DNA behind or carryover contaminants from the interphase and organic phase, lowering your overall DNA yield and quality. 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). DNA extraction. 4UYG$(. Plant DNA extraction is a hard, tedious and time-consuming process. The hazard with traditional CTAB protocols is the protein component of plant lysates is usually removed using phenol and chloroform. Ethanol and NaCl are used to remove plants polysaccharides. Centrifuge the sample at 25,000rpm for 5 to 8 minutes and transfer the supernatant into another tube. trailer Techniques covered include genomic DNA extraction using cetyl trimethylammonium bromide (CTAB) and chloroform extraction, chromatographic techniques, ELISA, hybridization, gel electrophoresis, dot blot analysis and methods for studying Not sterilized. 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. We've encountered a problem, please try again. Modification of CTAB protocol for maize. For the extraction and purification of DNA from leaves and seeds, cetyltrimethylammonium bromide (CTAB) is the "go-to" procedure. However, other homogenization such as rotor-stator homogenizer or bead mills can be suitable. 0000002963 00000 n After it becomes homogeneous, take the mixture into the 2ml Eppendorf tube. If someone says it, its wrong. Grow culture in 5 ml broth, pellet cells (~3000 g, 10 min), discard supernatant. Add 500 l 70% ethanol, resuspend pellet by flicking, allow to sit for ~5 min at room temp, then spin and drain again. Spin at top speed in microfuge (cold if possible) 10 min, drain off ethanol, retain pellet. 5- Washing . cold isopropanol. These impurities can also interfere with downstream applications, including PCR and NGS.10. 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. 3. other metals binds magnesium. DNA isolation methods are often modified and optimized for different cell types or sample sources. Extraction of DNA, RNA, and protein is the basic method used in molecular biology. DNA precipitates, resembling a thread of translucent white snot, at the interface between the juice and alcohol. zWf5 rGG]]no|/ 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. 2. This step also removes alcohol-soluble salt. polysaccharides in a high-salt environment. To keep up with this rapidly advancing field, DNA extraction protocols must be robust, flexible, consistent, and fast. 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. Precipitation. extraction method is a simple and non-toxic technique that isolates a high-quality DNA from the whole blood. Dellaporta (1983) and Maroof et al., (1984) have optimized the existing protocols and developed excellent methods for plant DNA extraction. 1.05K subscribers The CTAB method yields high quality plant genomic DNA that can be used for virtually any application including genotyping and Illumina sequencing. Vortexing steps can be replaced or supplemented by inversion and flicking of tube. Genomics Era for Plants and Crop Species Advances Made and Needed Tasks Ahead. uT`_zYduD=k isj`|p[!jhP9v7(v4x[P9 *n Mu^265!/f^P lNto5Yzs$xZ:4Qu.|fS=L;8cwX0 The DNA pellet do not dry and dissolved immediately in 300 l 1xTE, pH 8.0 at 55C for 5-10 minutes. For example, some plants need only simple grinding in mortar and pestle, while some need liquid nitrogen. However, care must be taken while handling liquid nitrogen. CTAB, cetyltrimethylammonium bromide, also called Cetrimonium bromide or hexadecyltrimethylammonium bromide. EDTA, the activity of present DNase is decreased. 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. iqy8D!fWJ64. of Red Blood Cells, RBC. Tap here to review the details. Euphrasia nankotaizanensis (Orobanchaceae) is a rare alpine herb that is endemic to Taiwan. The procedure for fixing the aptamer onto these transducers and for monitoring the interaction with the target protein is shown here. PowerPoint: Audio and Video Product Design: From Sketch to CAD . CTAB method is an organic solvent-based extraction method that has health hazards and harmful properties. Urea is yet another excellent lysis agent that can be used along and alone in DNA extraction. Romero Navarro JA, Phillips-Mora W, Arciniegas-Leal A, et al. Phenol-chloroform and isoamyl alcohol: 4. Find out more Dismiss 0000000893 00000 n CTAB is used in this method of DNA extraction. For example, cetyltrimethylammonium bromide (CTAB) and guanidium thiocyanate (GITC) are often included in protocols for DNA . Conclusively, PVP and beta-mercaptoethanol must be there in the plant DNA extraction buffer with the CTAB. Use PCI (phenol: chloroform: isoamyl alcohol) in combination with the CTAB buffer. In this case, after adding TE and NaCl to cell pellet, add lysozyme to 1 mg/ml, and incubate 1 hour at 37C. METHODS OF DNA EXTRACTION 1. 0000000812 00000 n Contrary, animal cells dont have turgor pressure, as it is not actually required. In this article, we will discuss plant DNA extraction and CTAB buffer. Add 500 l chloroform, vortex, spin 5 min, retain supernatant. After a considerable amount of time, the DNA may eventually float to the top of the alcohol layer. Steps in Organic and Inorganic DNA Extraction. additional purification, or wash, of the nucleic acid from the remaining salt. After grinding, the tissue is transferred to a tube and CTAB buffer is added. 70C, 1 hour). %%EOF Heating at 50-60C (up to an hour, intermittent mixing) and/or addition of more EB may be required to dissolve all the DNA. Instead of fuzzy bands, clear and sharp bands can be achieved by using a pinch of CTAB into the SDS PAGE. (100 mM Tris-HCl pH 8, 2% (w/v) CTAB, 20 mM EDTA, 1 M NaCl, and 1% (v/v) Main steps in DNA isolation procedure: 4 Lysis Removal of proteins and contaminants Recovery of DNA A number of commercial DNA purification kits use the same principles as the manual method.