The cetyl trimethylammonium bromide ctab method and its modifications have been used to obtain good quality total dna. Ctab cetyltrimethylammonium bromide plant dna extraction. The average total yield of maxwellextracted dna was comparable to ctab for the arabidopsis samples and was less than ctab, but higher than that of the dneasy method, for soybean and corn samples. The dna extraction kit1 is a modification of a procedure based on separating contaminating protein from dna by salt precipitation. Both dna extraction methods yield highpurity dna, but the modified ctab method yields higher amounts of dna than the other methods table 2, table a.
When ready to process sample, break sterivex following dna extraction protocol in blue notebook. The method may be used with either fresh or dehydrated plant material. The extraction of dna from seeds is a very important step in every molecular study involving plant genetics. When using the ctab method it takes at least 6 h to complete a dna extraction but our method takes 10 min. Structure of dna method of dna isolation maxwell 16 plant dna kit method spin column method ctab method reagent used in ctab method procedure for ctab method why dna extraction. Introduction ancient dna retrieved from postmortem bones and soft tissues provides scien. Isolating dna from plant tissues can be very challenging as the biochemistry between divergent plant species can be extreme. We have also used tissue prepared in advance by dessication. A simplified rice dna extraction protocol for pcr analysis 69 now relatively efficient and costeffective. Fungal dna extraction methods isolation of fungal dna is a multistep method. The methods were compared by the yield and purity of the extracted dna, its fragmentation state and suitability for amplification. Original article a simple method of genomic dna extraction. Nested pcr was shown to be a reliable approach to amplify ancient dna templates that failed in primary amplification.
The current extraction protocol is based upon the conventional cetyl. Comparison of the modified ctab to other dna extraction methods once all the favoured modifications to the selected lodhi et al. Cetyltrimethyl ammonium bromide ctab dna miniprep for. Although various conventional dna extraction protocols are now available, the process is still tedious, timeconsuming and needs toxic regents 78, e. Dna isolated by the ctabpvp method could be used in most molecular analyses. Figure 2 shows that, for all extraction methods the extracted dna performed well acceptable levels of qpcr inhibition in a qpcr assay. Simplification of dna extraction has been a major subject and as a result various methods are now available including commercial kits. In this article, we are going to discuss different types of dna extraction methods which are mostly used in genomic labs. Pdf ctab dna extraction method find, read and cite all the research you need on researchgate. Ctab extraction buffer effectively eliminates polysaccharides and polyphenols by employing the cationic detergent ctab. Dec 16, 2017 methods of dna extraction presented by.
Other studies used the dnazol methodology chomczynski et al. Many dna extraction protocols are known, including ctab and salt extraction method doyle and doyle, 1987 and its modification huang et al. Bromide ctab method, the proteinase k method and the chelex100 method, respectively. If the cells are in the early log phase, the culture should be placed on ice or 4c to slow down the growth and allow dna replication to complete prior to cell lysis and dna isolation. A simplified universal genomic dna extraction protocol. In this paper, in order to guarantee the quality and purity of genomic dna.
The method involves a modified ctab extraction, employing high salt concentrations to remove. Dna yields and purity of this method was then compared with the original method on various leaf. Dna extraction from different food matrices is a critical step in pcr analysis. Dna isolated with the dna extraction kit is free from contaminants and may be used directly for restriction digests, cloning, southern blotting, pcr amplification, and other dna analysis techniques. Ctab dna extraction buffer for plan dna extraction. A rapid and costeffective method for dna extraction from. Dna extraction ctab method we use this method for extracting genome sequencing quality i. Cells were resuspended in 500 l spheroplasting buffer plus 1% vv. A simplified rice dna extraction protocol for pcr analysis. Genomic dna extraction principle, steps and functions of.
Myriad permutations of the traditional phenolchloroform extraction methods e. Extraction of highquality genomic dna from different plant. Deionized water is used to get rid of ions which can interfere with the extraction and follow up pcr and other molecular analysis. An effective method for high purity genomic dna extraction. The pellet was resuspended gently in 500 l of extraction buffer, followed by the stepwise addition of. The results suggested that all three methods are effective in extraction of genomic dna of small insects. Unlike animal tissues where the same tissue type from different species usually have similar characteristics, plants can h. Pdf a modified ctab method for dna extraction from soybean. Existing methods use the neutral lysiscscl method or a dneasy blood tissue kit qiagen for dna extractions from liquid cultures gu et al.
The procedures assessed were the ctab method and wizard and qiagen dna. It was used to extract material for the micromonas rcc299 complete genome sequencing project, and the micromonas rcc472 genome sequencing project. Nov 30, 2009 3 ctab extraction method for plant extraction, the initial step that needs to be done is to grind the sample after freezing it with liquid nitrogen. Comparison of three genomic dna extraction methods to. The dna extracted by proteinase k and chelex100 protocols was too low to be detected on gel. Transfer supernatant to a new tube, care must be taken not to take any of protein pellet. Ctab based extraction buffers are widely used when purifying dna from plant tissues. Ctab buffer method, modified from plant dna extraction protocols, was found to have the highest success rate. Dna should be prepared from cell culture that is either in late log phase or early stationary phase. Different plant species have varying levels of polysaccharides, polyphenols, and other secondary metabolites which combine with nucleic acids during dna isolation and further affect the quality of the extracted dna. A simple method of genomic dna extraction suitable for. The aim of the present study was to explore the efficiency of a modified ctab method in comparison with three.
Reliable measurement of dna concentration and purity is important for almost all molecular genetics studies. Dna extraction using ctab lysis buffer and the traditional chloroformisoamyl alcohol purification step stewart and via 8. Comment in the comment box below and contribute to increasing the content of this article. Unlike animal tissues where the same tissue type from different. Pdf dna extraction ctab method phan thanh vy academia. Detailed pci dna extraction method, dna extraction method for plant tissue and for other tissues i will explain you in the next article. Different types of dna extraction methods are available for different cell types. Precipitated dna is washed with 70% ethanol, dried under vacuum and. For example, the dna extraction method for plant dna is different from that of the blood. Cetyltrimethyl ammonium bromide ctab is a surfactant useful for isolation of dna from tissues containing high amounts of polysaccharides.
An optimized ctab method for genomic dna extraction from freshlypicked pinnae of fern, adiantum. Extraction of highquality genomic dna from different. 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. Dna yields and purity of this method was then compared with the original method. The usual methods of dna extraction from filamentous fungi are either timeconsuming and require toxic chemicals or are based on expensive technologies cheng and jiang, 2006. Briefly, cell walls of fungal mycelia were broken down by grinding with glass rods or in the presence of liquid nitrogen. The present work describes an inexpensive ctabbased method with modifications for the extraction of highquality genomic dna from 19 different plant seeds. The dna from the grains of two maize hybrids, m10 and m321, was extracted using extraction methods dneasy qiagen plant mini kit, ctab method withwithout 1% pvp and modified mericon extraction. Thus, an alternative method that avoids chloroform has been developed by ops diagnostics. As a comparison to the thermolysis method, genomic dna was extracted with the established ctab method lee et al. Therefore, the modified ctab method could be the method of choice for dna extraction from complex and variously processed meat products and from raw and lowprocessed soybean. A comparison of different methods for preserving plant.
Dna is precipitated by the addition of room temperature isopropanol. Dna extraction by the ctab method as a comparison to the thermolysis method, genomic dna was extracted with the established ctab method lee et al. Moreover, total dna was extracted using ctab method and its4 and its5 primers were used for polymerase chain reaction pcr of rdna its sequences. Dna extraction dna extraction small scale using ctab method this method is relatively simple, and has been used successfully with a wide range of monocot and dicot species. An optimized ctab method for genomic dna extraction from.
A relatively quick, inexpensive and consistent protocol for extraction of dna from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Ctab method was efficient in the genomic dna extraction from single chironomid fig. What is the main purpose of ctabnacl in dna extraction. A simple and efficient genomic dna extraction protocol for. Dna quality and its performance in the qpcr assay were also monitored. Dna extraction optimisation a range of dna extraction procedures was assessed for the ability to efficiently and consistently extract both fungal and plant dna from diseased tissues. Dna extraction dna extraction is a procedure used to isolate dna from the nucleus of cells. The purpose of doing this step is to break down cell wall material of sample and allow access to nucleic acid while harmful cellular enzymes and chemicals remain inactivated. A simple and rapid dna extraction protocol for filamentous. Doyle and doyle 1987 were successful for dna isolation from small quantities of fresh leaf tissue using. However, for dna dissolved in te solution, the four samples with clear degradation after 36 months had all been extracted using the modified ctab method.
Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. Dna extraction from a sample is a process of purifying the dna. Exploring rapid and efficient protocol for isolation of. We use this method for extracting genome sequencing quality i. Protocols for extraction of dna of fungal cells are either very timeconsuming or show poor release of fungal dna5. Our study required extraction of dna from many samples and the dna quality had to be high enough to allow for pcrbased analysis such as ssr marker based genetic characterization table 3.
Modification of a ctab dna extraction protocol for plants. Ctab protocol for the isolation of dna from plant tissues. Dna influence the success of subsequent experiments, such as polymerase chain reaction pcr, enzyme digestion and recombinant selection. An efficient protocol for total dna extraction from the. Add the cut up filter membrane into a 2 ml mircocentrifuge tube with orange cap, trying to get the filter as close to the bottom as possible 4. Something to say about dna cell nucleus is the empire dna 4. But comparatively, the quality of dna sample isolated using the ctab method was better than those extracted using the other two methods, if the. Thus, these dna extraction protocols cannot be reproduced for all plant species porebski et al. We established a method that is simple, safe and fast compared to the ctab method which uses hazardous chemicals, is more time consuming and significantly expensive doyle and doyle, 1987.
We established a method that is simple, safe and fast compared to the ctab method. Genomic dna extraction with ctab cetyl trimethylammonium bromide notes. Genomic dna extraction principle, steps and functions of reagents 2. Polysaccharides and polyphenols are problematic contaminants associated with dna isolated from plants. Ctab extraction buffer is a widelyused reagent used to isolate dna from plant tissues. One option for purifying dna using ctab exploits that polysaccharides and dna have different solubilities in ctab. Figure 2 shows that, for all extraction methods the extracted dna. Therefore, the objective of present study was to develop a rapid, simple, lowcost and reliable method for dna extraction.
Cetyltrimethylammonium bromide ctab, sodium dodecyl sulfate sds, and qiagen kit qiagen methods were evaluated for extraction of genomic dna from young leaves suitable for pcr amplification for molecular analyses. Comparison of methods for dna extraction from a single. Ctab cetyltrimethylammonium bromide plant dna extraction medium to large scale from janice keller dnap, 6701 san pablo ave, oakland, ca 94608, u. Quantity and quality of the dna were examined spectrophotometerically and with gel electrophoresis. Dna was extracted using three methods listed below. Ctab extraction buffer effectively eliminates polysaccharides and polyphenols by employing the cationic detergent ctab hexadecyltrimethylammonium bromide or cetyltrimethylammounium bromide, and the. Dec, 20 thus, these dna extraction protocols cannot be reproduced for all plant species porebski et al. Something to say about dna cell nucleus is the empire dna. Under the highsalt conditions used in this protocol, the ctab. Several dna extraction procedures for isolating genomic dna from various plant sources have been described.
Dna must be purified from cellular material in a manner that prevents degradation. The cetyl trimethyl ammonium bromide ctab method and its modifications huang et al. Seeds possesses many compounds that effectively interfere with dna extraction. With high concentration of dna, this protocol provided lower od230od260 value and appropriate od260od280 ratio, which both were significantly different from 0. Use any protocol for dna precipitation, the one in this protocol works well. The sample can be tissue, plant or animal cells, blood, viral dna or any other dna containing sample. Ctab methods for dna extraction of sweetpotato for. Cetyltrimethyl ammonium bromide ctab dna miniprep for plant. This protocol can be used on most gramnegative bacteria to obtain high quality genomic dna, which is both pure and very high molecular weight. The cetyl trimethylammonium bromide ctab method and its modifications have been used to obtain good quality total dna for polymerase chain reaction pcr based downstream applications. One option for purifying dna using ctab exploits that polysaccharides and dna have different solubilities in ctab depending on the concentration of sodium chloride. When performing dna extraction there are three main general materialssolutions you will need. Pdf a modified ctab method for dna extraction from.
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