Genomic DNA Extraction Protocol Research Articles

High-Quality Genomic DNA Extraction Protocol for Bacillus and Clostridium Species.

High-quality DNA with sufficient yield is the goal of DNA extraction protocols. We present an optimized, cost-effective method for extracting next-generation sequencing (NGS)-quality genomic DNA from Bacillus and Clostridium species using the chloroform-isoamyl approach. The protocol involves two main procedures: cultivation of the bacteria under appropriate conditions, followed by DNA extraction through cell lysis, phase separation, DNA precipitation, and cleanup. This method has been successfully applied to several hundred strains of Bacillus and Clostridium species in our laboratory, including B. cereus, B. licheniformis, C. sporogenes, and C. tyrobutyricum, demonstrating its efficacy and reliability for producing high-quality DNA that meets NGS quality control standards. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing Bacillus and Clostridium species Basic Protocol 2: DNA extraction © 2024 by John Wiley & Sons, Inc.

Method for isolation of high molecular weight genomic DNA from Botryococcus biomass.

The development of high molecular weight (HMW) genomic DNA (gDNA) extraction protocols for non-model species is essential to fully exploit long-read sequencing technologies in order to generate genome assemblies that can help answer complex questions about these organisms. Obtaining enough high-quality HMW gDNA can be challenging for these species, especially for tissues rich in polysaccharides such as biomass from species within the Botryococcus genus. The existing protocols based on column-based DNA extraction and biochemical lysis kits can be inefficient and may not be useful due to variations in biomass polysaccharide content. We developed an optimized protocol for the efficient extraction of HMW gDNA from Botryococcus biomass for use in long-read sequencing technologies. The protocol utilized an initial wash step with sorbitol to remove polysaccharides and yielded HMW gDNA concentrations up to 220 ng/μL with high purity. We then demonstrated the suitability of the HMW gDNA isolated from this protocol for long-read sequencing on the Oxford Nanopore PromethION platform for three Botryococcus species. Our protocol can be used as a standard for efficient HMW gDNA extraction in microalgae rich in polysaccharides and may be adapted for other challenging species.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1pg/µl while root gDNA remained amplifiable at 10pg/µl. Genomic DNA obtained was also suitable for sequencing. This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Modified phenol chloroform protocol for genomic DNA extraction from keratinous tissue

DNA extraction enables the isolation and purification of genetic material by physical-chemical methods. There are specific protocols for different types of samples as animal and plant tissue. However, there are variations and particularities according to the nature of the fabric, such as the high presence of keratin. Thus, the present work aimed to develop and optimize a DNA extraction protocol from feather based on the Phenol-Chloroform method. Feather samples werw collected from 9 birds native to the Amazon region for the genetic material extraction. The DNA was precipitated with isopropanol and washed with 70% ethanol. Subsequently, the pellet was disolved in the TE buffer. The results indicated satisfactory concentrations for post-extraction molecular biology techniques such as PCR. The protocol showed a significant reduction in the incubation time to 1 hour compared to other protocols aimed at feather samples. Addition proteinase K to the lysis solution was the main factor that led to the optimization of the extraction time. The A260/230 and A260/280 absorbance ratios indicated the quality of the extracted DNA being close to what expected. Therefore, the results demonstrate the potential protocol use for molecular biology studies in birds from feather samples. However volume of phenol-chloroform and 70% ethanol can still be adjusted to suit the type sample.

Automation protocol for high-efficiency and high-quality genomic DNA extraction from Saccharomyces cerevisiae.

Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.

Comparative Study of Genomic DNA Extraction Protocols from Whole Blood for P53 Gene Polymorphism in Persons with and without Prostate Cancer

In latest decades, genetic methods have developed into a potent tool in a number of life-attaching applications. In research looking at demographic genetic diversity, QTL detection, marker-assisted selection, and food traceability, DNA-based technologies like PCR are being employed more and more. These approaches call for extraction procedures that provide efficient nucleic acid extraction and the elimination of PCR inhibitors. The first and most important stage in molecular biology is the extraction of DNA from cells. For a molecular scientist, the high quality and integrity of the isolated DNA as well as the extraction method's ease of use and affordability are crucial factors. The present study was designed to establish a simple, fast and inexpensive method for DNA extraction from human peripheral blood (normal male n=2, age 24 years old, patient male (prostate cancer) n=2, age 65 years old) by comparing between them, and aimed to standardize a protocol of DNA extraction using five extraction protocols. The first method was the modified organic method by using sodium perchlorate instead of organic solvent (phenol, chloroform), sodium perchlorate advantage comes from its cheap price and low storage and shipping requirements, the second was the enzymatic method by using proteinase K, third method was done by using detergent, the fourth used phenol-chloroform; finally fifth one was salting out method. The result showed that the organic method gives a good DNA yield and needs relatively short time while the enzymatic method gives an excellent DNA purity which are more suitable for PCR by comparing five protocols using the spectrophotometer and Nanodrop technetium in addition to electrophoresis. Through the use of the five suggested procedures, the PCR multiplication of the P53 gene with the isolated DNA was effectively carried out. This indicates that, with the exception of the detergent approach, there were no significant inhibiting substances for Taq polymerase in the final solution.

Evaluation of Easy and Economic Protocols for High Quality Genomic DNA Extraction and Sex Determination in Papaya Using SCAR Markers

Rapid and precise identification of three sex types (male, hermaphrodite and female flowers) is the key in achieving good returns in papaya (Carica papaya L.) cultivation. This study presents a simple, reliable, fast and cost-efficient DNA isolation protocol and a rapid and precise method of sex determination in papaya. Three DNA isolation protocols viz., CTAB method, Modified Dellaporta method and BioBasic DNA isolation kit were comparatively evaluated for their simplicity, economic, DNA quantity and purity. Among them, CTAB method was regarded as relatively economic, with a cost of Rs. 11.80943 per sample with higher amount of DNA (4667.4 µg/ µl). However, it was noticed that DNA isolation with the BioBasic kit was fastest method, which took only 6 hours 7 minutes. On the other hand, relatively better DNA purity was noticed with the Modified Dellaporta method (average ratio of absorbance at 260 nm and 280 nm range between 1.8-2.0) and yielded clear, intact and distinct DNA bands. Hence, Modified Dellaporta method is suggested as an ideal DNA isolation protocol for papaya. Molecular markers (such as SCAR derived from RAPD T12, W11, NAPF-2 and PKBT-4) were employed in this study to identify the sex type and they have successfully distinguished between male and hermaphrodite plants in both dioecious and gynodioecious varieties; especially, NAPF-2 has successfully distinguished male in dioecious varieties. Evaluation of different kinds of molecular markers has shown that the SCAR marker T12 can be used as reliable marker for rapid and precise identification of papaya sex type.

An improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity.

Shorea robusta (Dipterocarpaceae), commonly known as Sal, is an economically and culturally important timber species, known to contain a wide spectrum of polyphenols, polysaccharides, and other secondary metabolites in the tissues, which can interfere with the extraction of high-quality genomic DNA. In order to screen simple sequence repeat (SSR) markers and carry out other DNA-based analyses for this species in our laboratory, a high-throughput DNA extraction methodology was needed. Hence, we have optimized a simple, rapid, safe, and reliable high-throughput protocol for DNA extraction suitable for both fresh and dry leaves. The standardized protocol delivered good DNA yield of ∼1500 µg from 1 g of leaf tissue, with purity indicated by a 260 nm/280 nm absorbance ratio ranging from 1.70 to 1.91, which validated the suitability of extracted DNA and revealed reduced levels of contaminants. Additionally, the protocol that we developed was found to be suitable for polymerase chain reaction (PCR) amplification using microsatellite markers. Genome-wide characterization with SSR markers has been established in S. robusta, which further validates the protocol and its usefulness in DNA-based studies across the genus and/or family.

COMPARISON, VALIDATION, AND OPTIMIZATION OF INTERNAL GENOMIC DNA EXTRACTION PROTOCOL FOR CAMPYLOBACTER SPECIES

Campylobacter remains the leading cause responsible of human gastroenteritis worldwide. The current study aimed to compare the internal DNA extraction protocol with two commercially available kits, using C. jejuni (ATCC® 29428TM) and C. coli (ATCC®43478TM) and to validate and optimize the internal protocol through artificial contamination and confirmation of Campylobacter spp. from broiler chickens, turkeys, and beef meats samples. The extraction processes were carried out following the internal protocols steps and the manufacturer's instructions of PureLinkTM Genomic DNA Mini Kit and Wizard® Genomic DNA Purification Kit, respectively. The agarose gel electrophoresis system was used to control the DNA quality. After that, forty Campylobacter spp. isolates were confirmed, and finally, an artificial contamination of the aforementioned reference strains at three different concentrations was performed with sterilized minced meats. This is the first work comparing the PureLinkTM Genomic DNA Mini Kit and Wizard® Genomic DNA Purification Kit with internal genomic DNA extraction protocol for Campylobacter spp. The results indicated that the internal protocol provided similar efficiency to the two kits. All confirmed isolates were successfully amplified, in which28 isolates were C. coli and 12 were C. jejuni, as revealed by biochemical tests. A positive amplification was also observed in the three contaminated food matrices, after enrichment, at all examined doses. Except some reactions that were negative at 1 CFU/mL of C. jejuni and C. coli. This was explained by the detection limits of both internal protocol and qPCR. Based on our findings, three crucial steps in determining the extraction of DNA quality of this protocol were amended. Hence, the study highlighted the importance of validating simpler, cheaper and faster DNA extraction protocol, for each laboratory, as part of future risk assessment, control and monitoring programs of Campylobacter frequency required in molecular studies.

A rapid and inexpensive genotyping method using dried blood spots for mutational analysis in a mutant mouse model: an update.

Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a non-invasive sampling followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the specificity and feasibility of this novel procedure. Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a tiny amount of sample.

Comparison of two Genomic DNA Extraction Protocols from Single Dray Seed of Barley Crop

Modern plant breeding studies are largely based on plant genetic engineering programs. Extraction of DNA with high quality plays as a key factor in most of plant genetic Studies, therefore two different DNA extraction protocols based on CTAB buffer and SDS buffer were tested for the purpose of selecting the best DNA extraction protocol from dry seeds of nine variety of barely plant . Barley seeds were taken from Libyan seeds gene bank .After Barely samples were prepared DNA was extracted directly using CTAB and SDS solutions .The quality of extracted DNA was assessed by spectrophotometric measurements and gel electrophoresis system. The results of this study showed that purity of extracted DNA by CTAB method was clearly batter compared with SDS method. CTAB method seemed to be more effective for extracting DNA from barely dry seeds. High quality DNA obtained through use of CTAB method, while CTAB had overall better A260/A280 ratio (1.736-1.932). SDS method seemed to be not suitable for DNA extraction from dry seeds of barely crop.

Rapid and efficient protocol for genomic DNA extraction from leaf tissues of coconut (Cocos nucifera L.)

Good quality of the nucleic acid is the primary requisite for genomic research of crop plants. The presence of lipids, polysaccharides, polyphenols and protein molecules hinders downstream processes where genomic DNA has to be used as a template. Coconut leaf being highly fibrous and rich in all the secondary metabolites, isolation of good quality DNA remains a great challenge. Attempts to isolate the coconut DNA following the reported protocols are found not to yield DNA in the expected quality and quantity. A simple and fast approach for isolating the high-quality DNA from polysaccharides and polyphenolic-rich tissues of coconut is being detailed. As measured by its clear color, viscosity, and A260/280 ratio, the isolated DNA was devoid of polysaccharides, polyphenols, RNA, and other significant impurities. In addition to the detailing of the modifications made in the CTAB method, this paper discusses the major step-by-step improvements among the widely-followed DNA isolation protocols.

A Simple Method for Optimal DNA Extraction from Different Filamentous Fungi Species Growing on Earthen Walls of ‘Vale Histórico Paulista’, São Paulo, Brazil

ABSTRACT This study aims to describe a new simple and efficient method of extracting DNA from filamentous fungi isolated from historical earthen walls. This method involves growing the mycelium on overlapping cellophane discs on solid medium and the disruption of the fungal cell wall by a combined action of liquid nitrogen and a lytic enzyme. The extraction is performed with the PureLink ™ Genomic DNA Mini Kit protocol from Pure Genomic. The full procedure can be completed in two hours. The new genomic DNA extraction protocol provided good quality and quantity of fungal DNA, from fungal mycelium, with an average yield of 27 ng/µL of DNA per sample. Purified DNA samples yielded 25 ng of DNA ready for sequencing. PCR amplifications of the DNA extracted using the new method were more successful than those extracted using the CTAB method. Thus this new extraction protocol is demonstrated to be an effective method for the identification of a wide range of filamentous fungal species that affect the integrity of buildings with earthen architecture.

Standardization of protocol for genomic DNA extraction and microsatellite marker (SSR, ISSR) analysis in spine gourd (Momordica dioica)

The study evaluated reliable, easy and an efficient protocol for DNA extraction from young leaves of spine gourd which yielded highly pure with no visible discoloration concentrated genomic DNA ranged from 1202.68 to 3786.92 pg/500 mg of tissue with ratio (A260/A280) of absorbance ranging from 1.34 to 1.95. This method involved modification in original cetyl trimethyl ammonium bromide extraction without liquid nitrogen and by adding high level of P-mercaptoethanol, extracting twofold with chloroform: isoamyl alcohol (24: 1, v/v) and extending the centrifugation time which successfully removes polyphenols, chlorophyll pigments, polysaccharides and dyes. The DNA extracted by this method produced clearly scorable and reproducible definitive PCR fragments authentic exhibiting its compatibility and manifesting its affinity for SSR and ISSR markers. Therefore this method is recommended as an efficient protocol for genomic DNA extraction and microsatellite marker-based genetic analysis in spine gourd for high outp ut sample preparation for diverse PCR-based downstream applications.

Genetic diversity and comparative study of genomic DNA extraction protocols in Tamarix L. species

The genus Tamarix consists of about 54 species that mainly grow in saline areas of deserts and semi-deserts. This genus is chemically characterized by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. Thus it is necessary to optimize extraction protocols to minimize the influence of these compounds to the lowest level. The present study compares the efficiency of five different approaches to extract total genomic DNA in Tamarix species, showing significant differences in the extracted DNA contents and quality,by using Kit (DNP TM Kit), CTAB DNA extraction method by Murray and Thompson, Sahu et al., Nalini et al. and Bi et al., for the extraction of DNA from Tamarix species. Our results showed significant differences in DNA contents between these five methods. The quantity and quality of extracted genomic DNA were checked by the spectrophotometer, Nano-Drop and and agarose gel electrophoresis analysis. Finally, a PCR-based method was also applied to verify the amplification efficiency for two molecular markers (ITS and ISSR).. In the present study, the genetic diversity of 96 Tamarix individuals species and 8 populations were studied using 10 ISSR markerswhile for nrDNA ITS 8 species samples were used. The method of Nalini et al., provided best results (207 ng/μL) in terms of quantity and quality ofDNA. Our results proposed that this method could be effective for plants with the same polysaccharides, proteins and polyphenols components. The advantage of this method is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, and also do not requires expensive chemicals such as proteinase K, liquid nitrogen. ,. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Tamarix species group and the reliability of this method has been discussed.

P59.28 Protocol Adaptation for gDNA Extraction of FFPE Samples of Lung Adenocarcinoma Tissue and Related Mediastinal Lymph Nodes

Lung adenocarcinoma, the most common subtype of non-small-cell lung carcinoma (NSCLC), is characterized by low response to treatment with 50% rate of metastases at diagnosis and low five-year survival. To associate the molecular evaluation of mediastinal lymph nodes (MLN) obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to the histological examination could improve TNM staging and diagnosis. Our objective is to adapt and optimize the genomic DNA (gDNA) extraction protocol to be used in formalin-fixed paraffin-embedded (FFPE) samples from tumor tissue and mediastinal lymph nodes for molecular analysis. The gDNA from 28 paired samples of primary tumor (PT) obtained by transthoracic biopsy or tumor resection, and MLN obtained by EBUS-TBNA were extracted with GeneRead DNA FFPE kit (Qiagen). Fourteen paired samples were submitted to the manufacturer's protocol (MP- one 10 μm cut of FFPE samples incubated in deparafinization solution for 3 minutes at 56° C, followed by incubation in proteinase K, RNAse free water and FTB buffer for 1 hour at 56°C); eight paired FFPE samples were submitted to an adapted protocol (AP-no use of deparafinization solution, 3-4 10 μm cuts of directly incubated in a mix containing proteinase K, RNAse free water and FTB buffer for 3 hours at 56ºC), and 6 paired samples were submitted to both protocols (MP-AP). The gDNA quantification was performed in the Qubit 3.0 fluorometer (Thermo Fisher Scientific). Paired t test was applied, considering statistically significant p≤0.05. The range of gDNA yield obtained in the MP was 0.121-8.51 ng/μL for tumor samples and 0.025-1.14 ng/μL for MLN samples, and 1.04-26.0 ng/μL and 0.065-3.11 ng/μL, respectively, in the AP. In the paired samples extracted using both protocols, the range of gDNA obtained was 0.121-2.65 ng/μL and 1.04-4.95 ng/μL for PT samples (p=0.030) and 0.043-0.054 ng/μL and 0.065-3.11 ng/μL for MLN samples (p = 0.071). To obtain purified gDNA from FFPE samples is a laborious process, especially in archived samples over 5 years old and in samples with scarce cellularity, as are the small fragments of lung tissue obtained by transthoracic biopsy, or MLN fragments obtained by EBUS/TBNA. We observed a significant improving in the gDNA extraction for tumor samples using the AP protocol and a trend in improving for MLN samples, probably due to the small sample size. Changes in commercial protocols are frequently required to ensure better molecular results in these types of samples.

A Comparison of Genomic DNA Extraction Protocols in Artemisia annua L. for Large Scale Genetic Analyses Studies

A Comparison of Genomic DNA Extraction Protocols in Artemisia annua L. for Large Scale Genetic Analyses Studies

Optimization of DNA isolation and amplification protocol for Gracilaria and Sargassum species of Tamil Nadu coast

The present study proposes a simple and rapid protocol to isolate DNA from marine algae of the genus Sargassum and Gracilaria. To identify the easy, fast and low cost method, four methods of DNA isolation viz., The Robertson Labs methods, STE Buffer method, PureFast-Plant Genomic DNA purification kit by Helini Biomolecules, Chennai and DNA Isolation by Heat Lysis Method were chosen for the present study. To optimize the DNA isolation methods, the thalli of Sargassum plagiophyllum C. Ag. and Gracilaria salicornia C.Ag. were employed as a source of DNA. The DNA isolated based on three genomic DNA extraction protocols and by heat lysis method varied with reference to method employed for the isolation. By heat lysis method, the DNA of S. plagiophyllum and G. salicornia was isolated and simultaneously amplified (400 bp) using 23S rRNA gene primers. With these results, the DNA of G. salicornia, G. edulis, G. corticata, G. fergusonii, G. verrucosa, S. aquifolium, S. plagiophyllum, S. polycystum, S. tenerrimum and S. swartzii were isolated and amplified using heat lysis method. The heat lysis method is thereby proven to be efficient, rapid, simple, cost effective and requires only a small fraction of the sample. The heat lysis method can be a successful replacement for the conventional DNA isolation methods, when used specifically for DNA barcoding technique targetting organelle DNA.

A new, simple, highly scalable, and efficient protocol for genomic DNA extraction from diverse plant taxa.

PremiseCommonly used molecular techniques such as next‐generation sequencing require reliable methods to extract DNA quickly and efficiently. Secondary compounds within plant tissues make this requirement all the more challenging, often forcing researchers to test different extraction methods tailored to their particular species of interest in order to obtain large amounts of high‐quality genomic DNA. The opportunities provided by high‐throughput, next‐generation sequencing only exacerbate these problems, especially when trying to extract DNA from multiple species at the same time. Several methods have attempted to resolve the challenges of obtaining suitable DNA from plants; however, a rapid, high‐yield, high‐quality, and highly scalable DNA extraction method is still needed.Methods and ResultsWe present a rapid DNA extraction protocol that utilizes a buffer with relatively large amounts of cetyltrimethylammonium bromide (CTAB) and sodium chloride, combined with a silica maxi‐column cleanup of the extracted DNA. The new method is easy to implement using standard equipment and inexpensive reagents. The entire procedure (from grinding to the final elution) can be completed in less than two hours for a single sample and can be easily scaled to meet desired research goals. It works on diverse green plants with highly varied secondary chemistries (e.g., ferns, gymnosperms, and phylogenetically divergent angiosperms).ConclusionsApplication of the protocol to various plant species yielded DNA of high quality in less than two hours and can be adjusted to extract DNA at large (maxi‐preps) or small (96‐well minipreps) scales. We anticipate that our method will be of wide utility for rapidly isolating large quantities of quality genomic DNA from diverse plant species and will have broad applications in phylogenetic studies utilizing PCR and short‐read DNA sequencing.

Optimization of genomic DNA extraction protocol for black wattle

Considerando-se que a atual tendência do melhoramento florestal é a integração das técnicas clássicas com as de análise genética molecular, faz-se necessária a obtenção de protocolos de extração de DNA genômico ajustados a cada espécie estudada. O objetivo do trabalho foi determinar o efeito de diferentes adaptações no protocolo de extração de DNA genômico CTAB para acácia-negra. Foram testados diferentes componentes na fase de extração orgânica: clorofórmio, fenol e proteinase K, além da aplicação de RNase após a fase de precipitação e limpeza do DNA. Também, foi investigada a eficiência destes tratamentos em amostras de folíolos frescas ou armazenadas em baixa temperatura durante sete dias. Foi verificada a presença de DNA de todas as amostras submetidas à extração pelo protocolo de CTAB com os diferentes tratamentos. O tempo de armazenamento das amostras não influenciou na integridade do DNA, entretanto, foi possível observar que a adição de RNase melhorou a qualidade do DNA extraído. Deste modo, sugere-se a utilização do protocolo CTAB com uso de clorofórmio e RNase, com amostras frescas ou armazenadas em baixas temperaturas.