The mantra to success in plant DNA extraction is to grind tissue well, hard, and rough until fine homogenization. Contrary, animal cells dont have turgor pressure, as it is not actually required. pH 8.0 Autoclaved. Plenty of proven DNA extraction chemicals are available, you can choose according to your need. Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet. CTAB BUFFER 500 ml-1 140mM Sorbitol 12.8 g 220mM Tris, pH 8 55 ml of 2M 22mM EDTA 22 ml of 0.5M 800mM NaCl 80 ml of 5M 1% Sarkosyl 5 g 0.8% CTAB 4 g Combine, check pH = 8, autoclave. Sorry, preview is currently unavailable. 0000001496 00000 n 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? By accepting, you agree to the updated privacy policy. Plant cells can be lysed with the ionic detergent CTAB, which forms an insoluble complex with CTAB is used in this method of DNA extraction. PRESENTED BY: AYESHA KABEER Magnetic bead-based DNA extraction: 8. Sterile Eppendorf tubes and desired reagents. membranes (such as those around the mitochondria and chloroplasts) have been broken Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Get news, product info, tips, industry updates, events, freebies, and more delivered right to your inbox. inactivation of cellular nucleases and separation of the desired genomic DNA from cellular DNA extraction by chromatography: 2. Not sterilized. The solid phase protocol listed below is an alternative. zWf5 rGG]]no|/ DNA extraction Method for plant sample using CTAB method. 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} } Now wash the DNA pellet with alcohol two times at 9000 rpm for 2 minutes. Bromide (CTAB) protocol (developed by Murray and Thompson in 1980) is appropriate for the out, while the nucleic acid precipitates. 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. 4UYG$(. Use the manufacturers guide for use. You have to modify the protocol and preparation as per your need. Afterward, the homogenized sample would be incubated with lysis buffer and protease enzyme for effective lysis of the cell wall. DNA extraction. The nucleic acid is separated and precipitated using alcohol followed by a few rounds of washing with 70% ethanol. trailer Tannin is actually the main culprit. t>V nk+ex!J};0R6B7wRR: %]?i=UXW0l4uRvREu?*js\2cWa>#T|n}%VV?WyuT"8}r6a}s8mP*bL {iWMs(U /M_]{VGZ"\+ Cell wall and nuclear membrane lysis- by chemical treatment, tissue homogenization using liquid nitrogen and physical grinding. Genomic DNA was extracted using the CTAB method (Doyle & Doyle, 1987), with some modifications, including a Sorbitol pre-treatment to remove mucilaginous polysaccharides (Souza & al., 2012). Autoclave all the utilities prior to use including solutions. Activate your 30 day free trialto unlock unlimited reading. 2'md%^Zgr~ {7">Jf-"{JK#QKyD50o(GCsEwh]j48Ljbl@tmJ 'A5zQvKC*?Zpp9sm1/;hfk Cell lysis is aided with a CTAB buffer, which also stops secondary metabolites from obstructing DNA extraction. Nederlnsk - Frysk (Visser W.), Applied Statistics and Probability for Engineers (Douglas C. Montgomery; George C. 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CTAB* is a strong detergent, it will damage eyes and irritate skin. x\KsFW1XD$I~,5>x,}HeVVwmk-m?X/VW|\ Grind the tissue in a mortar and pestle and add Urea buffer (which contains Urea, Tris, EDTA and NaCl). extraction and purification of DNA from plants and plant derived foodstuff and is particularly Fast ID contains powerful denaturants and DNA-selective This paper is only a guide for these techniques and we describe. To keep up with this rapidly advancing field, DNA extraction protocols must be robust, flexible, consistent, and fast. Once the tissue has been frozen, its ground into a fine powder with a mortar and pestle or a blender. EDTA is a chelating component that among dissolved in the aqueous solution are separated from the lysis homogenate. Spin at top speed in microfuge (cold if possible) 10 min, drain off ethanol, retain pellet. The distribution of alpine herbs is severely threatened by climate change, which influences genetic variation and population structure. 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. Use PCI (phenol: chloroform: isoamyl alcohol) in combination with the CTAB buffer. 2. DNA isolation methods are often modified and optimized for different cell types or sample sources. For this Magnesium is a cofactor for DNase. *n1cFPw There are no repetitive and lengthy phase separation steps or hazardous reagents used, so you can further streamline your labs operations and protect the safety of key personnel. Muhammad I, Zhang T, Wang Y, et al. Stabilizing and precipitating DNA- by chemical treatment using NaCl and alcohol. Besides, the turgor pressure is another factor that we have to look after. 0000001976 00000 n Start with sample preparation, clean the tissue and prepare it for homogenization. 10 Secret Tips for DNA Extraction to Get Good Results. [2] CTAB binds to the polysaccharides when the salt concentration is high, thus removing polysaccharides from solution. 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. However, other homogenization such as rotor-stator homogenizer or bead mills can be suitable. UC - Davis Safety Services website: https://safetyservices.ucdavis.edu/safetynet/safe-use-of-phenol. The following stock solutions and reagents were prepared by mixing the standard amount of chemicals. Application of Genome Wide Association and Genomic Prediction for Improvement of Cacao Productivity and Resistance to Black and Frosty Pod Diseases. <]/Prev 62123>> 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). ^ 0=pLRto6Q&N(Na*Y;"I$1k*wkX 5|AQ>4DWii/:&"E N So you do not need to waste the time on rewritings. Clipping is a handy way to collect important slides you want to go back to later. Sterile pestle and mortar XeQ CTAB, cetyltrimethylammonium bromide, also called Cetrimonium bromide or hexadecyltrimethylammonium bromide. Free access to premium services like Tuneln, Mubi and more. Furthermore, our protocol has been optimized to work with a wide range of plant species and sample types, enabling novel and rapid advancements in modern plant genomics. Beta-mercaptoethanol can irritate upon inhalation. High molecular weight DNA yield in the range of 328 to 4776 ng/L with an average . QLnoSc3 TuAWbAJ7$ (5hiHFn%wCEJ}=PbcSB#hN(YYXl69Dr ]\79/SiSM J,QOY5#^A@r|)(' g@9_d[v9|L.Th*9I5D(Y Click here to review the details. Notes from Kazakh Khanate till Political Dynamics + reading answers, Solutions - 6th Edition Computer and Networking, Stock Watson 3U Exercise Solutions Chapter 5 Instructors, A sample of letter of enumerator addressed to your employer, The Love Hypothesis Chapter 16 Adams POV by Ali Hazelwood (z-lib, Business Statistics and Mathematics Solved Past Papers, B.com Part 1 Punjab University 2009-2018, Siki soku 104 - dg sdg sdg fdg dsf sdg sdg sdg fdg dsf sdg sdg sdg fdg dsf sdg sdg sdg fdg dsf. 0000000812 00000 n 6- Dilute . 70C, 1 hour). Use of phenol/chloroform also generates organic waste which requires special storage containers and disposal procedures. Despite the existence of a huge collection of Thermococcales strains, very few plasmidic elements are reported so far in the hyperthermophilic euryarchaeotes. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA. with ice cold Ethanol. 0000002394 00000 n 1. The first is a small (3.5 kb) cryptic plasmid found in a high copy number in the marine species P. Add liquid nitrogen to the tissue and roughly grind the sample into the mortar and pestle. Besides its role in DNA extraction, CTAB is used as an antiseptic (against bacteria and fungus), detergent and ingredient of cosmetics. After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE. 22. The precipitate is pelleted by centrifugation and washed with 70% ethanol to remove salts introduced during extraction. Add 500 l chloroform, vortex, spin 5 min, retain supernatant. However, other chemicals like RNase, proteinase K, SDS and PCI may be used to improve the quality and yield of DNA. Wash with around 700 L of 70% ethanol. In addition, plant DNA extraction is even a difficult task to execute because of their specialized cell structure and composition. After the cell and organelle Avoid using 2 or more days-old CTAB solutions. Add the beta-mercaptoethanol just before the use. Weve updated our privacy policy so that we are compliant with changing global privacy regulations and to provide you with insight into the limited ways in which we use your data. 0000000536 00000 n (1991) used 0.5% SDS with the CTAB buffer and developed a simple plant DNA extraction protocol. Summary Aptamers are an alternative to antibodies in their role as biorecognition elements in analytical devices. Prepare a 10X stock for CTAB solution to avoid repeated chemical preparation. If that all seems like a bit much, you arent wrong. Bl~oNiefef_ L>{$^bKb~dh~REBtz(4>/kP',2"32GnF(8xi9 Overdrying of the final DNA pellet is BAD. EDTA, the activity of present DNase is decreased. This protocol is my own, we used it in our labs during our masters. This protocol originally came to us from Evelyne Derelle at Observatoire Ocanologique, Banyuls sur Mer. 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. EB wont give as much protection as TE, which also protects against nucleases. Extracted DNA of rose flowers using Cetyl Trimethyl Ammonium Bromide (CTAB) with three different solvents to study the method that gives maximum yields of DNA. Grow culture in 5 ml broth, pellet cells (~3000 g, 10 min), discard supernatant. edu/Mstats/. Remember to boil RNAse before use to kill any DNAse in it (see Sambrook for method). ice-cold ethanol (1000 l) to precipitate DNA incubate at -20C for at least 1 hour. Under these IntechOpen website: https://www.intechopen.com/chapters/49877. However, the CTAB-like buffer system can do better with such hard tissues. Liquid nitrogen (if available) 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. DNA precipitates, resembling a thread of translucent white snot, at the interface between the juice and alcohol. Major ingredients of CTAB buffer are CTAB, Tris, EDTA, beta-mercaptoethanol, PVP and NaCl salt. In a high salt (NaCl) concentration, the detergent forms an METHODS OF DNA EXTRACTION 1. Notedly, keep in mind that the composition of the buffer and concentration of chemicals would differ from plant species to species. Precipitating the DNA with an alcohol usually ice-cold ethanol or isopropanol. v;K6n8>0xx~fzNFLscB=)lo-s7iW:QxzA+ o45bW`=bN htt& In the CTAB procedure, the first step is breaking down the tissue, and it involves freezing your plant sample using liquid nitrogen. Salting-out method. Conclusively, PVP and beta-mercaptoethanol must be there in the plant DNA extraction buffer with the CTAB. Liquid nitrogen can burn the skill and even do permanent damage. It actually challenges our knowledge to optimize things in our lab. Aboul-Maaty NAF, Oraby HAS. Sterile spatulas (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 I will also explain variations and concentrations of different CTAB buffers that you can try and use. The beta-mercaptoethanol remains in the supportive role, but effectively blocks the oxidation of tannin, thereby disallowing interaction with DNA. 3. 0000000016 00000 n p|`ylk/?|+.NB/;3a"a~w Y"nX#%C %Ss`2*;2%R=aX&~-o%LZax [C? ?MlB`>8O`-51H>tr$Tt=eyP Le|\! Euphrasia nankotaizanensis (Orobanchaceae) is a rare alpine herb that is endemic to Taiwan. Solid-liquid phase DNA extraction: 10 Different DNA extraction methods: 1. 2.8. Various chemical constituents of DNA extraction buffer allow effective isolation. %%Jb4jgAgluFd&Hw4[hply_.FnZL1ywbIWuB}^zl|pq. CTAB DNA extraction is a simple, effective, cheap and reliable method to extract DNA from plants, especially for RAPD analysis and barcoding. DNA extraction is a process of isolating 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. 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 chloroform denatures the proteins and facilitates the. 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 . J w9K|F>.-c#0G{3F)2jSV7\snE>rl gCe:"6zkO( But differences in the cell structure and chemical composition of a cell require a different DNA extraction setup. Removing proteins by adding a protease (optional but almost always done). 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. Why is DNA extraction from plants difficult? 4- DNA . Results from six grams of leaf tissue finely ground using a mortar and pestle, then aliquoted (1 g) for each extraction. Plant cell walls are very difficult to break down and the cells contain many compounds that impede extraction and inhibit downstream molecular biology applications. Separation step (Extraction of Protein) Organic DNA Extraction: PCI. %PDF-1.3 We've encountered a problem, please try again. Take 100 mg tissue, homogenate << /Length 1 0 R /Filter /FlateDecode >> It is a major constituent of polyphenolic compounds and is almost present in all plants. 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. A fragment analyzer Qsep100, that uses a capillary gel electrophoresis system, allowed . Ideal lysis procedure is rigorous enough to disrupt the complex starting material (plant In this study, we demonstrate a modified CTAB method for the isolation of DNA to maximize the quality and yield from small aphids. Murray MG and Thompson WF. ii. Only four small populations remain in Xue, Nanhu, and Cilai Mountains of Taiwan. recovery of dna from agarose gel, rajendra prasad central agricultural university. 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. Cornejo OE, Yee MC, Dominguez V, et al. 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. Basic steps for DNA extraction 3. 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. 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. Along with CTAB, chemicals like SDS and PVP gave an excellent yield for Corn and Soybeans. 4 0 obj Care must be taken while handling CTAB as it has corrosive and toxic properties. RNase A can be added to remove RNA either add this at the start of the prep, or to the dissolution buffer used at the end of the prep. Modified DNA extraction protocol i. Preheat the 3 extraction buffer in water bath at 65 C. The CTAB buffer facilitates cell lysis and prevents secondary metabolites from interfering with DNA extraction and downstream procedures. CTAB Protocol for the Isolation of DNA from Plant Tissues. 1. Precipitation. (ii) Add 25 ml CTAB buffer and transfer to 50 ml tube. startxref Once the nucleic acid complex has been 0000000893 00000 n UV absorbance spectrometry. other metals binds magnesium. Research . PK ! 10% w/v CTAB (Cetyl trimethylammonium bromide), Autoclaved. In principle, in the extraction process, CTAB associates with the cell wall polysaccharides and proteins and co-precipitates it along with DNA. Hae9 ?jvo-nc(lu-~yiV+o=}==~4m5g&>wwce-onKa2J{xUsR!~}wTatTk U+kZBwGo-TNb> (Xqu%n h=vi% It instantly froze tissue below its freezing temperature and eventually ruptures the cell wall. Former website http://biogeek. (100 mM Tris-HCl pH 8, 2% (w/v) CTAB, 20 mM EDTA, 1 M NaCl, and 1% (v/v) 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. What is the Role of Alcohol in DNA extraction? of Red Blood Cells, RBC. A micro-level optimization is required to use it for our use. I already have explained the different types of polysaccharides and polyphenols present in plants and required chemical modifications accordingly. We've updated our 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. Use 70% ethanol to wash DNA pellet. Do not autoclave alcohol and phenol. suitable for the elimination of polysaccharides and polyphenolic compounds otherwise affecting 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. 5. The salting-out method is a non-toxic DNA extraction method described by Miller, Dykes, and Polesky in 1988. Increasing the heat and duration of the CTAB step might help with tough cells (eg. CTAB solution: 3% CTAB, 1.5 M NaCl, 10 mM Na 3 EDTA, 0.1 M MOPS-acid; Dr Tushar Chauhan is a Scientist, Scientific-writer and co-founder of Genetic Education. Discard all waste phenol and chloroform into chemical waste bottle in fume hood. DNA quality was checked on 1% agarose gels using a ladder and the purity was checked as above. By binding Mg with CTAB extraction buffer. Be sure you have the proper safety protocols in place before starting your DNA extractions. choosing a selection results in a full page refresh. 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. Percentages are represented as w/v. The SlideShare family just got bigger. ), and the procedures can be done in microfuge rather than big centrifuge (faster!). The results were qualitatively equivalent in all methods. After a considerable amount of time, the DNA may eventually float to the top of the alcohol layer. Weve updated our privacy policy so that we are compliant with changing global privacy regulations and to provide you with insight into the limited ways in which we use your data. Report on Carcinogens, Fourteenth Edition. Leaf samples of two diseased and two asymptomatic plants were collected, and DNA was extracted using a modified CTAB DNA extraction method (Murray & Thomson, 1980). For example, some plants need only simple grinding in mortar and pestle, while some need liquid nitrogen. K6-0001-050 or -200) is designed for processed and/or multi-ingredient foods. 2. 0 DNA extractions using a traditional CTAB-based method with (A) no PVP, (B) 1% PVP and (C) 4% PVP. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. National Toxicology Program website: https://ntp.niehs.nih.gov/ntp/roc/content/profiles/chloroform.pdf. To achieve more consistent tissue disruption across samples, you can also use a blender, though this step is still low throughput and time consuming. Add 500 l chloroform, vortex and mix thoroughly, incubate on ice 30 min. 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. 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. The CTAB procedure would likely work with many gram-negative strains without modification, but gram positives would likely require the addition of lysozyme and proteinase K steps, and may also require modification of medium eg. Plant DNA extraction using CTAB is simple and effective. Free access to premium services like Tuneln, Mubi and more. Vortexing steps can be replaced or supplemented by inversion and flicking of tube. Add liquid nitrogen and grind vigorously and add a lysis buffer, simultaneously. In this case, after adding TE and NaCl to cell pellet, add lysozyme to 1 mg/ml, and incubate 1 hour at 37C. These are essential for maintaining and further supporting the rapidly evolving pace, scope, and scale of agricultural R&D. Total genome DNA from samples was extracted using the CTAB/SDS method . Do you know the CTAB can also be used in the SDS-PAGE? Genomics Era for Plants and Crop Species Advances Made and Needed Tasks Ahead. Use proteinase K overnight at room temperature or for 2 hours at 60C. 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 . You can also skip the lengthy RNase digestion, incubation and centrifugation periods, and precipitation steps. Slowly pipette out and retain supernatant avoid interface material and lower phase. Importantly, follow a common lab safely and perform the experiments using liquid nitrogen under the supervision of experts. polysaccharides in a high-salt environment. NaCl. 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. Nguyen Quoc Trung. Proteinase K DNA extraction: 6. Conclusion: Heat may be used to extract DNA from K . 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. How To Extract DNA From Whole (Fresh), Frozen, Dried and Clotted Blood? stream For teachers that have done DNA extraction from strawberry or thymus, fewer cells collected in this experiment. To make powder with liquid nitrogen or crush it in sterilized pestle mortar with 2 mL of Other chemicals required in the process are SDS, protease enzyme, Phenol, chloroform, isoamyl alcohol and ethanol. homogenate to sterile Eppendorf tube.
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