A cheap,reliable and rapid method of extracting high‐quality DNA from plants

Here we describe a rapid method for extracting DNA from plant material using a microtitre plate system. This extraction procedure has been tested using three species, Brassica napus, Brassica napus/rapa hybrids and Arum maculatum, stored frozen, in silica gel or used fresh. The length of storage was between 7 days and 2 years, and in all cases high molecular weight DNA was reliably purified. Polymerase chain reaction (PCR) testing, using multiplex and single product amplification, inter simple sequence repeats (ISSRs) and microsatellites, was always reliable. This method combines the speed of commercial 96‐well plate methods with the economies associated with readily available laboratory chemicals.     

A high-throughput DNA extraction protocol for tropical molecular breeding programs

Liquid handling robotics and capillary electrophoresis genetic analyzers now offer high-throughput solutions for 2 of the 4 key steps in PCR-based DNA marker-assisted fingerprinting (DNA extraction, PCR amplification, electrophoresis, data analysis). Thus, DNA extraction remains the most significant bottleneck at the bench for large-scale applications in plant breeding and germplasm characterization. We report on a rapid and low-cost method for relatively high-throughput extraction of high-quality DNA from young and mature leaves of sorghum, pearl millet, chickpea, groundnut, and pigeonpea. The procedure uses a modified CTAB/β-mercaptoethanol method for DNA extraction in a 96-well plate. The quantity and quality of the DNA extracted per sample is adequate for more than 1000 PCR reactions. A relatively high throughput of 96–384 samples per person per day can be achieved, depending on the crop. A major timesaving aspect of the protocol is the absence of a manual sample-grinding step. Finally, the cost is a magnitude lower than commercial plate-based kits, and, as such, is likely to have substantial application in tropical molecular breeding programs.     

一种冬虫夏草全基因组DNA的提取方法

冬虫夏草是真菌与昆虫形成的复合生物体,本研究建立了一种可同时提取冬虫夏草真菌子座和虫体全部基因组DNA的方法。该方法稳定高效,简便易行,提取纯度高,适用于冬虫夏草多重PCR、Realtime-PCR和DNA指纹图谱等分子水平的研究。     

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at −80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.     

A high-throughput plant DNA extraction method for marker analysis

The use of molecular markers to improve crops depends on the availability of rapid and efficient DNA extraction methods. Here we describe a simple and inexpensive method to isolate plant DNA suitable for RFLP, AFLP, and simple sequence repeat (SSR) analysis. This procedure uses stainless steel ball bearings to grind 16 samples simultaneously using a high-speed flask shaker. The method used in routine laboratory exercises yields 120–144 DNA extractions in a day by a single person at a cost of $0.60 (AUD) per sample, doubling the throughput of conventional methods.     

A fast and inexpensive DNA extraction/purification protocol for brown macroalgae

Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < €0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated.     

一种经济高效提取实蝇基因组DNA的方法

单头实蝇高质量基因组DNA的获得为实蝇分子生态学研究奠定了重要基础。本文提出一种经济高效的实蝇基因组DNA提取方法,该方法广泛适用于不同虫态、不同保存条件的实蝇标本,与传统的CTAB法和SDS法相比操作简单、耗时短,得率高。     

木本植物基因组DNA提取及鉴定

采用改良后的CTAB法,对山葡萄、软枣猕猴桃、蒙古栎、核桃楸、西伯利亚红松和偃松基因组DNA进行提取。结果表明,所提基因组DNA分子量与λDNA（48 kb）接近,其紫外吸收比在1.66～1.89之间。第3次和第4次上清提取的DNA质量优于第1次和第2次。从提取产量看,每克鲜重提取DNA量最小为15 μg·g^-1（核桃楸第4次上清）,最高的为272 μg·g^-1（山葡萄第3次上清）。西伯利亚红松和偃松第1次和第2次上清基本未提出DNA,第3次和第4次上清中得到了较高质量的DNA。经酶切鉴定和PCR扩增,所提的基因组DNA可以用于进一步研究。     

An inexpensive high-throughput method to extract high yields of good quality DNA from fungi

We developed an efficient method for high-throughput extraction of high-quality DNA from various fungi. In this method, fungal mycelia were cultured and harvested on the surfaces of membranes on media plates. We degraded cell walls using a lytic enzyme (Yatalase). Purification was performed on 96-well glass fibre filter plates. DNA was successfully extracted from various fungi provided (102 genus 132 species) at high yields and quality, and proved suitable for storage, polymerase chain reaction amplification and restriction enzyme digestion. The method described is rapid, inexpensive and automation friendly. This enables the simultaneous extraction of large numbers of samples, significantly improving the potential throughput in genomics, particularly in diagnostic and population studies.     

A protocol for high-throughput extraction of DNA from rice leaves

Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less than 6 mo of storage at 4°C.     

Combining bleach and mild predigestion improves ancient DNA recovery from bones

The feasibility of genome‐scale studies from archaeological material remains critically dependent on the ability to access endogenous, authentic DNA. In the majority of cases, this represents a few per cent of the DNA extract, at most. A number of specific pre‐extraction protocols for bone powder aimed to improve ancient DNA recovery before library amplification have recently been developed. Here, we test the effects of combining two of such protocols, a bleach wash and a predigestion step, on 12 bone samples of Atlantic cod and domestic horse aged 750–1350 cal. years before present. Using high‐throughput sequencing, we show that combined together, bleach wash and predigestion consistently yield DNA libraries with higher endogenous content than either of these methods alone. Additionally, the molecular complexity of these libraries is improved and endogenous DNA templates show larger size distributions. Other library characteristics, such as DNA damage profiles or the composition of microbial communities, are little affected by the pre‐extraction protocols. Application of the combined protocol presented in this study will facilitate the genetic analysis of an increasing number of ancient remains and will reduce the cost of whole‐genome sequencing.     

A simplified protocol for preparing DNA from filamentous cyanobacteria

The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations, and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants.     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

A modified mini-prep method for economical and rapid extraction of genomic DNA in plants

Molecular markers for map-based cloning, marker-assisted selection in crop breeding, and genetic studies require DNA isolation from a large number of plants in a short span of time. Here we describe a modified DNA extraction method that is economical in terms of cost, time and labour. The method allows DNA extraction from as little as 0.2–0.3 g of leaves that are homogenized in zipper plastic bags, followed by DNA isolation in 1.5-mL Eppendorf tubes. By using the modified method, a DNA yield of 700–800 μg/300 mg leaf tissue was obtained from cotton and wheat samples. The quality of the DNA was quite suitable for PCR-based markers.     

A cost-effective,simple and high-throughput method for DNA extraction from insects

Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 μL-deep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present.     

Review of methodologies for extracting plant-available and amorphous Si from soils and aquatic sediments

There is a variety of methodologies used in the aquatic sciences and soil sciences for extracting different forms of Si from sediments and soils. However, a comparison of the published extraction techniques is lacking. Here we review the methodologies used to extract different Si fractions from soils and sediments. Methods were classified in those to assess plant-available Si and those to extract Si from amorphous silica and allophane. Plant-available Si is supposed to comprise silicic acid in soil solution and adsorbed to soil particles. Extraction techniques for plant-available Si include extractions with water, CaCl₂, acetate, acetic acid, phosphate, H₂SO₃, H₂SO₄, and citrate. The extractants show different capabilites to desorb silicic acid, with H₂SO₃, H₂SO₄ and citrate having the greater extraction potential. The most common extractants to dissolve amorphous silica from soils and aquatic sediments are NaOH and Na₂CO₃, but both also dissolve crystalline silicates to varying degrees. In soils moreover Tiron is used to dissolve amorphous silica, while oxalate is used to dissolve allophanes and imogolite-type materials. Most techniques analyzing for biogenic silica in aquatic environments use a correction method to identify mineral derived Si. By contrast, in the soil sciences no correction methods are used although pedologists are well aware of the overestimation of amorphous silica by the NaOH extraction, which is most commonly used to extract silica from soils. It is recommended that soil scientists begin to use the techniques developed in the aquatic sciences, since it seems impossible to extract amorphous Si from soils completely without dissolving some of the crystalline silicates.     

A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants

Protocols for genomic DNA extraction from plants are generally lengthily, since they require that tissues be ground in liquid nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening projects.     

Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD)

A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).