buffering capacity (a low or high pH damages DNA). DNA extraction Method for plant sample using CTAB method. See how we can help you extract high-quality plant DNA, in less time. Cell preparation and extraction techniques. Major ingredients of CTAB buffer are CTAB, Tris, EDTA, beta-mercaptoethanol, PVP and NaCl salt. JuMRUN.)oMf91 E@4E+t5mAc5fJHI3?K#Sk[~Php#3D t
jhPd|c|4bHVOQE%M%Zh**W0o-B 0ZN&1 Breaking cells open to release the DNA. abyssi strain GE5. In this article, we will discuss plant DNA extraction and CTAB buffer. 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. This protocol is my own, we used it in our labs during our masters. INORGANIC METHOD OF DNA EXTRACTION
3. EDTA, the activity of present DNase is decreased. Manufacturers recommend homogenization of samples with mortar and pestle in presence of liquid nitrogen. 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. Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.). In this study, we investigated the effects of the natural isolation of alpine habitats . 2 0 obj The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA. In the standard salting-out method, proteins K and RNase are added to them after the lysis of cells. Steps in Organic and Inorganic DNA Extraction. Note also that all waste chloroform and phenol arising from this procedure must be discarded as hazardous chemical waste (not down the sink!). 2. To keep up with this rapidly advancing field, DNA extraction protocols must be robust, flexible, consistent, and fast. Basic steps for DNA extraction 3. (C++) the answers of assignment ( c++, 2nd edition), Cmo activar Office 2019 gratis y sin programas, Assignment 1. Let me explain to you in brief, turgor pressure is water pressure that occurs from inside the cell to the cell wall. I already have explained the different types of polysaccharides and polyphenols present in plants and required chemical modifications accordingly. Summary Aptamers are an alternative to antibodies in their role as biorecognition elements in analytical devices. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. 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. 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. )Z$m|h_8"dOtv#P%w;:K&z]x.A`5.AZ%q9;eU! 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. 37 0 obj
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[2] CTAB binds to the polysaccharides when the salt concentration is high, thus removing polysaccharides from solution. Plan your day carefully and set aside the proper amount of time to complete the entire protocol. Tannin is actually the main culprit. (1991) used 0.5% SDS with the CTAB buffer and developed a simple plant DNA extraction protocol. 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. Aboul-Maaty NAF, Oraby HAS. EXTRACTION (i) Grind 2 to 5 g of frozen leaves to a very fine powder using N2 cooled mortar and pestle. 4. %PDF-1.4
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Liquid nitrogen (if available) By whitelisting SlideShare on your ad-blocker, you are supporting our community of content creators. Grind the tissue in a mortar and pestle and add Urea buffer (which contains Urea, Tris, EDTA and NaCl). 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. Find out more Dismiss CTAB extraction buffer, the detergent captures the lipids and the proteins allowing the For example, some plants need only simple grinding in mortar and pestle, while some need liquid nitrogen. We use this method for extracting genome sequencing quality (i.e. The structure is explained in the figure below. Saturated NaCl was needed for the proteins to precipitate out of the solution. 0000000536 00000 n
Add 1/10 volume Na-acetate (50 l) and 2 vol. _:)a"P$,4+ 5>15Kzv$& Caution must be taken when working with liquid nitrogen for the first grinding step as it can rapidly freeze skin tissue and cause cold burns even with short exposure. MATERIALS: ^ cQmG;Q5h7~S.KC10NdXwkx|%!UPvDPXN2_82q?Lz>O&=pq7^9vmA(PdLe m C1!j 3SK97vU|(TgEw'q>jh:k"a6==UDnMW+6i4~t5F&CfYn*OGB"GrL#~]mT$zUIa 6/iDbe h'*omlb_i*G'*MBP q08C q~x]ufAi=Y
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Enter the email address you signed up with and we'll email you a reset link. METHODS OF DNA EXTRACTION Safe Use of Phenol | Safety Services. ), and the procedures can be done in microfuge rather than big centrifuge (faster!). Contrary, animal cells dont have turgor pressure, as it is not actually required. hb```Lf| Care must be taken while handling CTAB as it has corrosive and toxic properties. %%Jb4jgAgluFd&Hw4[hply_.FnZL1ywbIWuB}^zl|pq. What is the Role of Alcohol in DNA extraction? Be sure you have the proper safety protocols in place before starting your DNA extractions. The lysis of the membranes is accomplished by the detergent However, care must be taken while handling liquid nitrogen. PK ! Chloroform: Mixture of chloroform and isoamyl alcohol (24:1 ratio) equilibrated with a layer of TE buffer (pH 8) on top. Plants bear secondary metabolites and a diversely rigid cell wall made up of polysaccharides like pectin, cellulose, chitin, etc. 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. method, providing an overview before delving deeper into the procedure in a step-by-step approach. | DNA Extraction Method 2. Discard all waste phenol and chloroform into chemical waste bottle in fume hood. The DNA pellet do not dry and dissolved immediately in 300 l 1xTE, pH 8.0 at 55C for 5-10 minutes. 6R(D$2(>1bD44b9!nGH-NCgVw
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As a result, you may leave DNA behind or carryover contaminants from the interphase and organic phase, lowering your overall DNA yield and quality. %%EOF
Various chemical constituents of DNA extraction buffer allow effective isolation. May need to be heated to 50C to dissolve. 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. Ch05 - Chapter 05 solution for Intermediate Accounting by Donald E. Kieso, Jerry J. 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. 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. x0pnzmM )3jo.o8e'nn/j!;+RRRJ8F9k^ iN Qfe=\%A9BL
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In addition, plant DNA extraction is even a difficult task to execute because of their specialized cell structure and composition. You can download the paper by clicking the button above. aqueous solution with chloroform. Now customize the name of a clipboard to store your clips. Gw.:S /vR.fP"
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54o)IR pZ|h!0"# CTAB buffer (preheated at 65C for 15 min.) other metals binds magnesium. Muhammad I, Zhang T, Wang Y, et al. Extraction of DNA from Infected Cassava and Banana from Cameroon . Honey is a natural product widely consumed all over the world due to its relationship with healthy benefits. 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. Dr Tushar Chauhan is a Scientist, Scientific-writer and co-founder of Genetic Education. It was created in the 1980s, and since then, several adjustments have been made for usage with . x$)FcD2k@ c@5#3Q`e^KDzqjpcm|;lm+ou>oyu?mei{:.||vtz~mG18zh]w \v?o.?;]wWv:auaw:t@-Og8ArNg(quz|JFv\'nC~w^P2uwpvRwghEPC"w;x@wb:^Q$~P9&*RlJAWfb:wT]8PJ\*u;Tut7.TJOD$F:ZTi/=l6QMoT
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if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[336,280],'geneticeducation_co_in-leader-2','ezslot_9',168,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-leader-2-0'); No universal CTAB DNA extraction protocol exists, keep in mind this thing. . Euphrasia nankotaizanensis (Orobanchaceae) is a rare alpine herb that is endemic to Taiwan. How To Extract DNA From Whole (Fresh), Frozen, Dried and Clotted Blood? the DNA purity and therefore quality. Store final DNA preparation at -20C. 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. Under these conditions, proteins, phenolic The Cetyl Trimethyl Ammonium Removing proteins by adding a protease (optional but almost always done). We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. Not sterilized. 0000003202 00000 n
Besides its role in DNA extraction, CTAB is used as an antiseptic (against bacteria and fungus), detergent and ingredient of cosmetics. mix gently by inverting and spin at 13,000 rpm at 4C for 10 min. K to 0.5 mg/ml, incubate 60C 1-16 h). Spin at top speed in microfuge (cold if possible) 10 min, drain off ethanol, retain pellet. It was used to extract material for the Micromonas RCC299 complete genome sequencing project, and the Micromonas RCC472 genome sequencing project. RNA aptamers, specific for different proteins, have been exploited as biorecognition elements to develop specific biosensors (aptasensors). This protocol originally came to us from Evelyne Derelle at Observatoire Ocanologique, Banyuls sur Mer. Dissolve in 50 L Wash cell pellet in TE (resuspend then pellet again) before lysis if nuclease activity is a problem. Cell wall and nuclear membrane lysis- by chemical treatment, tissue homogenization using liquid nitrogen and physical grinding. 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. For example, cetyltrimethylammonium bromide (CTAB) and guanidium thiocyanate (GITC) are often included in protocols for DNA . Method of DNA extraction: Many different methods and technologies are available for the isolation of genomic DNA. In 1980, Murray and Thompson proposed an inexpensive and simple protocol for plant DNA extraction by CTAB. sufficiently purified, precipitation can be accomplished. ii. additional purification, or wash, of the nucleic acid from the remaining salt. Click here to review the details. if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[250,250],'geneticeducation_co_in-leader-1','ezslot_23',145,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-leader-1-0'); Homogenization is a process to prepare a homogeneous mixture of plant tissue using tissue homogenization techniques like physical grinding using a Mortar and pestle. 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. Nonpolar molecules migrate into the organic phase and leave behind DNA and other polar molecules in the aqueous phase. However, the CTAB-like buffer system can do better with such hard tissues. DNA extractions using a traditional CTAB-based method with (A) no PVP, (B) 1% PVP and (C) 4% PVP. 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? We've updated our privacy policy. After the clear pellet appears, add 500L of TE buffer or elution buffer to the pellet and dissolve the DNA in it. The nucleic acid is separated and precipitated using alcohol followed by a few rounds of washing with 70% ethanol.
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h=vi% A fragment analyzer Qsep100, that uses a capillary gel electrophoresis system, allowed . CTAB method is an organic solvent-based extraction method that has health hazards and harmful properties. Modification of CTAB protocol for maize. A micro-level optimization is required to use it for our use. If youre 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. w;owwllND\| 2. 5. Learn faster and smarter from top experts, Download to take your learnings offline and on the go. CTAB* is a strong detergent, it will damage eyes and irritate skin. JP`N @$;(O2@%/n&73PLK`K!:x &P1N{Yrp}r>\XpY3_,(\! After the cell and organelle insoluble complex with the polysaccharides. polysaccharides in a high-salt environment. DNA was diluted to 1 ng l 1 with sterile distilled water. step (Lysis of White blood cells, WBC) 3- Phase . Alex: Gee, thats a lot of chloroform, might be a while before I risk trying this protocol. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added. Separation is also successful when the tissue contains high amounts of polysaccharides. 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. Importantly, follow a common lab safely and perform the experiments using liquid nitrogen under the supervision of experts. The solid phase protocol listed below is an alternative. Academia.edu no longer supports Internet Explorer. It is a cationic detergent, and water and alcohol soluble. Various chemical constituents of DNA extraction buffer allow effective isolation. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. t>V nk+ex!J};0R6B7wRR: %]?i=UXW0l4uRvREu?*js\2cWa>#T|n}%VV?WyuT"8}r6a}s8mP*bL {iWMs(U
/M_]{VGZ"\+ It was adapted from Winnepenninckx B. et al., 1993, TIG: 9 (12), 407 (Technical Tips). SDS (sodium dodecyl sulfate) is an excellent anionic detergent that can lyse proteins. (100 mM Tris-HCl pH 8, 2% (w/v) CTAB, 20 mM EDTA, 1 M NaCl, and 1% (v/v) Nucleic acids research. 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. 5. CTAB is used in this method of DNA extraction. You choose the most appropriate method ! tissue), yet gentle enough to preserve the target nucleic acid. Discard pellet and take supernatant in another tube, Wash the sample with the alcohol (minimum 2 wash). [*ZeJ3"P|7He ]nBx)rgrRSfh3sVPkYQxs811Bwiw"gML
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Inorganic DNA Extraction: 6M . The chloroform denatures the proteins and facilitates the. Spin at ~10,000 g in microfuge (cold if possible) for 10 min. Pre-prepare a CTAB solution (10%) by heating and store it at 37C temperature. To make things even more effective use RNase as well in combination with proteinase K. Proteinase K and RNase are commercially available. In general, plant DNA extraction and purification can be divided into six steps: 1) tissue disruption/homogenization, 2) cell lysis in DNA extraction buffer, 3) separation of DNA from other cellular components, 4) DNA precipitation, 5) DNA washing, and 6) DNA collection/resuspension for downstream processing. Then the amount of phenol and chloroform used is greatly reduced (you can use 500 l instead of 5 ml ! However, in practice, the protocol has several drawbacks: its lengthy, tedious, and low throughput, with many steps that require careful handling, exposure to hazardous chemicals, and several other technical considerations. DNAITPCRDNA of Red Blood Cells, RBC. 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. The method is fast,. These are essential for maintaining and further supporting the rapidly evolving pace, scope, and scale of agricultural R&D. 0000002394 00000 n
Removal of contaminants and other cell debris- by chemical treatment using SDS, CTAB, PVP, beta-mercaptoethanol and Triton X 100, etc. different method, providing an overview before delving deeper into the procedure in a step-by-step approach. >,xPD/-}V62h
(s['HXGF=_fCUVY~ .ET_Xwk6A.xYkNMaR^+C?x'"LhaxDU|&O'Z extraction and purification of DNA from plants and plant derived foodstuff and is particularly If you will be using it a lot, consider making multiple smaller aliquots, since repeated freeze/thawing will deteriorate the quality. and nucleus wall. 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. 1980; 8(19); 43214325. 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. Former website http://biogeek. Ch13 - Chapter 13 solution for Intermediate Accounting by Donald E. Kieso, Jerry J. 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. Liquid nitrogen can burn the skill and even do permanent damage. Redissolve pellet in 100 l EB. This paper is only a guide for these techniques and we describe. Heating at 50-60C (up to an hour, intermittent mixing) and/or addition of more EB may be required to dissolve all the DNA. membranes (such as those around the mitochondria and chloroplasts) have been broken PRESENTED BY: AYESHA KABEER It will surely make things easy. Washing and elution- By alcohol and TE (Tris-EDTA) buffer. Precipitating the DNA with an alcohol usually ice-cold ethanol or isopropanol. They work in a similar fashion, apart from CTAB being a cationic detergent while SDS is anionic. The more finely your tissue is ground, the more efficient you DNA extraction will be, making this a critical step for successful DNA extraction. In addition, the efficiency of PVP increases greatly by adding beta-mercaptoethanol. Murray MG and Thompson WF. conditions, the detergent i. CTAB, which is more stable in alcohol than in water, can be washed METHODS OF DNA EXTRACTION
1. However, TE can interfere with subsequent enzyme reactions (EB wont). Free access to premium services like Tuneln, Mubi and more. Mix vigorously and incubate in water bath at 65C for 1 hr. EB (Elution buffer): 5 mM Tris-HCl. Do not sell or share my personal information. Chloroform is toxic by inhalation and will also damage eyes. uT`_zYduD=k isj`|p[!jhP9v7(v4x[P9 *n
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lNto5Yzs$xZ:4Qu.|fS=L;8cwX0 The aqueous phase is collected, and the recovered DNA is precipitated out with isopropanol. 1. Additionally, environmental and ethical issues have a higher weight in the consumer's choice to buy honey as a natural product. choosing a selection results in a full page refresh. << /Length 1 0 R /Filter /FlateDecode >> The distribution of alpine herbs is severely threatened by climate change, which influences genetic variation and population structure. A proteinase K step could be added at the same point as the lysozyme step, or immediately after the lysozyme step (add prot. We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. It appears that you have an ad-blocker running. Molecular genetic techniques are an effective monitoring tool, but high-quality DNA samples are usually required. Liquid nitrogen prepares the fine power of tissue and also deactivates nucleases by providing an extra chill environment. I .g$!=n]BQ|nTr|0T5t4@XIR$fwyVIszC9Er(ze*RzoI$M%8FM=Mi fR{X[:[F R=XFBdvk>*H7&NIP'B.4GsXUS6j'^p2+#hY[xBYUJuk
(L)4/vB`])tS2\YI!MSCKVN",NAN}/DKHFR and transfer the powder or 600 L of Tap here to review the details. CTAB BUFFER 500 ml-1 140mM Sorbitol 12.8 g 220mM Tris, pH 8 55 ml of 2M 22mM EDTA 22 ml of 0.5M 800mM NaCl 80 ml of 5M 1% Sarkosyl 5 g 0.8% CTAB 4 g Combine, check pH = 8, autoclave. the pellet Bromide (CTAB) protocol (developed by Murray and Thompson in 1980) is appropriate for the p|`ylk/?|+.NB/;3a"a~w Y"nX#%C %Ss`2*;2%R=aX&~-o%LZax [C? ?MlB`>8O`-51H>tr$Tt=eyP Le|\! 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. (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 .
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