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1.
A modified mini-prep method for economical and rapid extraction of genomic DNA in plants 总被引:1,自引:0,他引:1
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. 相似文献
2.
目的建立并评价FTA-DNA直接提取法在病原真菌分子鉴定中的应用。方法采用whatman FTA-DNA直接提取法从25个不同种属的45株培养的菌株和6例临床标本中提取病原真菌DNA,用于病原真菌的测序鉴定。配制不同浓度的孢子悬液探索该方法的检测限和安全性。结果 45株菌株扩增后均能得到1条清晰的DNA扩增片段,并成功测序。应用该方法亦成功从腹水、胸水、口腔拭子、宫颈拭子来源的临床标本中直接提取DNA并成功鉴定病原真菌。该DNA提取方法联合降落PCR能检测到1.0×103个cell/mL的孢子悬液,1.0×104个cell/mL及以下浓度的孢子悬液可以被FTA卡完全灭活。结论 FTA-DNA直接提取法可快速有效地从培养的菌株及部分临床标本中提取并保存病原真菌DNA,用于病原真菌的测序鉴定。 相似文献
3.
A simple and reliable method for extracting DNA has been developed for orchid species and hybrids. The high quality of DNA
obtained is suitable for amplification via the polymerase chain reaction (PCR) for producing random amplified polymorphic
DNA (RAPD) markers.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
4.
A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue 总被引:18,自引:0,他引:18
We describe an inexpensive method for dehydration of plant tissue and extraction of high molecular weight DNA. Tissue is dried
for 12 to 24 hours in a food dehydrator and subsequently powdered for DNA extraction. Dicot tissue can be powdered in centrifuge
tubesen masse using a commercial paint mixer and glass beads. With the use of the paint mixer, tissue never touches common surfaces that
might lead to cross contamination, a potential benefit when the DNA is to be used for PCR reactions. The DNA is of a quality
equal to that obtained from either lyophilized or fresh frozen tissue (commonly used in many labs). The advantages of the
described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations
where large numbers of samples must be extracted. 相似文献
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Josquin F. G. Tibbits Luke J. McManus Antanas V. Spokevicius Gerd Bossinger 《Plant Molecular Biology Reporter》2006,24(1):81-91
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. 相似文献
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Bertini L Amicucci A Agostini D Polidori E Potenza L Guidi C Stocchi V 《FEMS microbiology letters》1999,173(1):239-245
The alignment of the 28S gene of several species of Pezizales allowed to select two pairs of primers able to amplify the internal transcribed spacer region of ribosomal DNA in mycorrhizal fungi, such as truffles. The higher yield of the amplification product demonstrates a better annealing of the new primers to the rDNA, as compared to the universal primers internal transcribed spacer 1 and internal transcribed spacer 4. Therefore, the new primers can be used as an easier and more sensitive tool for the identification of truffle species in any stage of their life cycle, including the mycorrhizal phase. 相似文献
10.
一种快速提取肠道微生物总DNA的方法 总被引:3,自引:2,他引:3
采集的兔肠道内容物及其粪便样品,通过分散浸泡、震荡洗涤、分级离心、滤器过滤、DNA提取试剂盒提取纯化,可以获得纯度很高的DNA样品。经0.8%琼脂糖凝胶电泳检测和紫外分光光度计测定,样品A260/A280的比值为1.72±0.02。分别以提取的DNA样品为模板,通过设计的细菌特异引物,对其16S rDNA基因进行PCR扩增,获得了1.6 kb大小特异性很好的预期条带。这为肠道微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。 相似文献
11.
An intensive survey was carried out on a 12-year-old experimental truffle bed of Tuber melanosporum Vitt. located in the central
Apennines. The aim of the investigation was to relate the presence and carpophore production of T. melanosporum to changes
in soil structure, aeration and fertility — expressed in terms of 0.25–2.00 mm aggregate fraction, total organic carbon, DTPA-extractable
Mn and host plant height — and to determine if these modifications, whenever present, could be ascribed to soil differentiation
within the truffle bed. The occurrence of pianelli — i.e. areas with little herbaceous ground cover created by T. melanosporum
— showed a close relationship with host plant height and aeration of soil surface layers. Where pianelli occurred, the height
of symbiont trees increased and the content of reduced Mn, indicating the presence of a well-aerated soil environment, decreased.
The variation of host plant height was attributable not only to the increased absorption of nutrients related to the ectomycorrhizal
partnership, but also to soil differentiation. The soils of the investigated area were characterized by a relatively low slope
gradient, a rigid framework of gravel and a homogeneous physico-chemical behaviour, due to the predominance of Ca among exchangeable
bases. In these environmental conditions, T. melanosporum was present in the rather thick soil belonging to Typic Rendolls,
whereas it was absent in the area characterized by thin Lithic Rendolls. In the latter case, the plant cover was probably
too scarce to protect T. melanosporum from summer dryness, and consequently the more resistant T. aestivum species prevailed.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
12.
Xinping Zhao Hong-Bin Zhang Rod A. Wing Andrew H. Paterson 《Plant Molecular Biology Reporter》1994,12(2):110-115
A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps,
(i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7
Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning. 相似文献
13.
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. 相似文献
14.
A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species 总被引:2,自引:2,他引:0
Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality,
restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems. 相似文献
15.
Xia Liu Yongdong Xu Zhi Li Shengwei Jiang Shuo Yao 《Preparative biochemistry & biotechnology》2018,48(4):378-382
A silica sands-based method has been developed to isolate high quality genomic DNAs from cells of animals, plants and microorganisms, such as Hemisalanx prognathus, Spinacia oleracea, Pichia pastoris, Bacillus licheniformis and Escherichia coli. To the best of our knowledge, no DNA isolation method has so wide application until now. In addition, this method and a commercially available kit were compared in analysis of microbial communities using high-throughput 16s rDNA sequencing. As a result, the silica sands-based method was found to be even more efficient in isolating genomic DNA from gram-positive bacteria than the kit, indicating that it would become a very valuable choice to faithfully reflect the composition of microbial communities. 相似文献
16.
L. BERTINI D. AGOSTINI L. POTENZA I. ROSSI S. ZEPPA A. ZAMBONELLI & V. STOCCHI 《The New phytologist》1998,139(3):565-570
Five species of white truffle were classified using PCR-based techniques. RAPD (random amplified polymorphic DNA) fingerprints and specific pairs of primers were used. A RAPD fragment was constant in Tuber borchii Vittad. isolates and polymorphic among the other species. Two molecular markers specific for T. borchii were developed from the sequence of the non-polymorphic RAPD fragment and from regions flanking the 5'-3' ends of a truffle gene. These markers were applied in the identification of T. borchii fruit bodies, mycelia and mycorrhizas, allowing us to monitor the development of this fungus during its entire life cycle. 相似文献
17.
Biochemical and molecular characterization of NADP-glutamate dehydrogenase from the ectomycorrhizal fungus Tuber borchii 总被引:2,自引:1,他引:1
Luciana Vallorani Emanuela Polidori Cinzia Sacconi Deborah Agostini Raffaella Pierleoni Giovanni Piccoli Sabrina Zeppa Vilberto Stocchi 《The New phytologist》2002,154(3):779-790
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Genetic analyses using museum specimens and ancient DNA from fossil samples are becoming increasingly important in phylogenetic and especially population genetic studies. Recent progress in ancient DNA sequencing technologies has substantially increased DNA sequence yields and, in combination with barcoding methods, has enabled large-scale studies using any type of DNA. Moreover, more and more studies now use nuclear DNA sequences in addition to mitochondrial ones. Unfortunately, nuclear DNA is, due to its much lower copy number in living cells compared to mitochondrial DNA, much more difficult to obtain from low-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples and at the same time allows processing many samples within a short time frame is immediately required. In fact, the major bottleneck in the analysis process using samples containing low amounts of degraded DNA now lies in the extraction of samples, as column-based methods using commercial kits are fast but have proven to give very low yields, while more efficient methods are generally very time-consuming. Here, we present a method that combines the high DNA yield of batch-based silica extraction with the time-efficiency of column-based methods. Our results on Pleistocene cave bear samples show that DNA yields are quantitatively comparable, and in fact even slightly better than with silica batch extraction, while at the same time the number of samples that can conveniently be processed in parallel increases and both bench time and costs decrease using this method. Thus, this method is suited for harvesting the power of high-throughput sequencing using the DNA preserved in the millions of paleontological and museums specimens. 相似文献
20.
Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield. 相似文献