共查询到20条相似文献,搜索用时 15 毫秒
1.
Jing Ning Jost Liebich Matthias Kästner Jizhong Zhou Andreas Schäffer Peter Burauel 《Applied microbiology and biotechnology》2009,82(5):983-993
Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits,
we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as
the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient
gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities
of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected
by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due
to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until
now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced
by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based
methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the
subsequent assessment of microbial community. 相似文献
2.
Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation. 相似文献
3.
Techniques for purifying the purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromoprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.0.3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation. 相似文献
4.
Purified preparations of watermelon curly mottle virus (WCMoV), a whitefly-transmitted geminivirus, contained dimeric or geminate particles of 20 times 30 nm and the virus was transmissible by mechanical means. Virus yields ranged from 100–150 μg/100 g leaf tissue. Purified preparations exhibited a typical nucleoprotein absorbance profile with a maximum absorbance at 258 nm, and A280 / A260 ratio of 0.61–0.64. Infectivity was associated with two light-scattering, virus-containing bands following sucrose density gradient centrifugation. The viral capsid protein was resolved as a doublet by SDS-PAGE. The estimated mol. wts of the two bands within the doublet were 29 100 (±1550) and 27 733 (±1550), respectively. DNA isolated from virus particles was resolved by gel electrophoresis into two circular single-stranded DNA bands of approximately 2.6 to 2.7 kb. The two bands are believed to represent the individual components of a bipartite genome, characteristic of previously described whitefly-transmitted geminiviruses. 相似文献
5.
6.
The diaminobenzoic acid dihydrochloride fluorescence method has been examined to determine optimal conditions for the assay of DNA in perchloric acid extracts and in pretreated Chlamydomonas cells on glass-fiber filters. Maximal fluorescence yield was obtained by allowing: (a) the perchloric acid extract to react with 4–6% diaminobenzoic acid dihydrochloride for 20–40 min at 60–80°C at about pH 3.0; (b) the pretreated cells on glass-fiber filters to react with a 20% solution of diaminobenzoic acid dihydrochloride for 40 min at 60°C. 相似文献
7.
Moeller R Horneck G Rettberg P Mollenkopf HJ Stackebrandt E Nicholson WL 《Current microbiology》2006,53(3):227-231
RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore
germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical
density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the
ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/108 spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from
dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple
to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was
a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically
decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however,
the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores
is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental
conditions. 相似文献
8.
A circular dichroism technique for the determination of DNA base composition in terms of the guanine-cytosine content has been developed. A linear relationship between the ratio of ellipticities at 260 and 280 nm (theta 260/theta 280) and base composition of DNA is obtained in citrate-phosphate buffer, pH 3.0 at 20 degrees C. The DNA base composition obtained with this method is not only highly reproducible but is also the fastest method reported. 相似文献
9.
This paper describes a protocol effective at extracting high yields of high-purity microbial community DNA from humified soils. DNA was extracted from soil by lysozyme, SDS and freeze–thaw lysis, precipitated and then subjected to a double caesium chloride density gradient centrifugation stage before concentrating and washing. Evaluation using three soils yielded up to 30 μg DNA g−1 dry soil, with absorbance ratios at 260 : 230 nm and 260 : 280 nm of 1·6–2·0. The DNA extracted from the three soils was digested by four restriction enzymes and a 16S rDNA eubacterial product was amplified by PCR. These tests indicated that the DNA obtained by the protocol was sufficiently pure for molecular biological analysis. 相似文献
10.
一种简单有效的植物RNA提取方法 总被引:25,自引:0,他引:25
在提取缓冲液中加入皂土有效地去除了蛋白质并抑制RNAse,建立了一种高效的植物RNA提取方法。以麻疯树幼叶为材料,分别用TRIZOL,异硫氰酸胍法,SDS-KAc法和新创皂土法提取总RNA进行比较和验证,琼脂糖凝胶电泳和紫外光谱分析结果表明,只有皂土法能提出质量高,完整性好的RNA。进一步以皂土法提取的18S rRNA为模板的RT-PCR结果分析表明, 用该法提取的RNA 能用于分子克隆与基因表达分析等后继分子生物学实验。该方法简便,快速,不失为一种经济高效的RNA提取方法。 相似文献
11.
从500g新鲜牛肝制得蛋白质二硫键异构酶(PDI,EC 5.3.4.1)98mg。该酶制剂在SDS-聚丙烯酰胺凝胶电泳中表现为亚基分子量62,000的均一条带。在260nm追踪,因二硫键错接而失活的牛胰核糖核酸酶A,经PDI作用使其二硫键重排恢复活力,从而催化酵母RNA的水解来测定PDI活力。这种单波长法比文献中介绍的追踪A_(260)—A_(280)的双波长法更为灵敏方便。酶的克分子消光系数ε_M=1.03×10~5(pH7.5),其比活性为1400单位/克蛋白质。 相似文献
12.
High-quality plant DNA extraction for PCR: an easy approach 总被引:1,自引:0,他引:1
I. Ahmed M. Islam W. Arshad A. Mannan W. Ahmad B. Mirza 《Journal of applied genetics》2009,50(2):105-107
Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies.
For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and
efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires
maceration of plant tissue of about 1.0 cm2 (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes,
followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether
is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm−2 for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR
using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers
by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato
and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability
of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive. 相似文献
13.
Natasha M. Sanabria Melissa Vetten Charlene Andraos Kailen Boodhia Mary Gulumian 《PloS one》2014,9(12)
Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220–340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190–220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190–220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects. 相似文献
14.
Sera of mice immunized with ribosomal fractions of Candida albicans showed the presence of anti-C. albicans antibodies, detected by the gel-immunodiffusion, agglutination and immune adherence tests.Candida infections are among the most prevalent opportunistic yeast infections, attacking debilitated individuals, and against which there is no effective prophylactic treatment currently available (1, 2, 3, 4, 5). In view of the succes reported in experimental immunizations with ribosomal fractions from various bacteria and some fungi, as summarized by Youmans and Tewari (7, 8), a similar approach for immunization in experimental candidiasis appears reasonable. The present work describes preliminary results on circulating antibodies elicited in the course of immunizations with ribosomal fractions of Candida albicans.Ribosomal preparations were obtained from mechanically disrupted cell-pellets of C. albicans by differential centrifugation and purification in a 15% sucrose and 5% ammonium sulfate solution (in sodium-magnesium-Tris buffer), using a modification of the procedure described by Rubin (6). Concentration of ribosomal-RNA was determined by the absorbance at 260 nm; ribosomal-protein concentration by the Lowry reaction; and purity of the ribosomal preparation checked by the ratio of absorbance at 260 nm to 280, and at 260 to 235 nm. Mice (ICR strain) were immunized with these ribosomal preparations in amounts of 50–100 g ribosomalprotein/mouse, by 2–3 subcutaneous inoculations with Frend's adjuvant, with a 10–21 day interval between the inoculations.This work constitutes part of Ruth Levy's research study towards the Ph.D. degree. 相似文献
15.
Mechanical Transmission, Purification and Properties of an Isolate of Maize Stripe Virus from Venezuela 总被引:2,自引:0,他引:2
A Venezuelan isolate of maize stripe virus (MStpV) was successfully transmitted mechanically and by the leafhopper Peregrinus maidis from field infected plants to sweet cv. Iochief. After purification of maize infected with MStpV, fine spiral filamentous particles about 4 nm in diameter and with variable lengths were consistently associated with a nucleoprotein band present in CsCl or Cs2SO4 isopycnic gradients. Purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 260–263 nm and a minimum at 240–243 nm and a 260–280 ratio of 1.38. The density of the nucleoprotein in CsCl gradients was estimated at 1.29 g/ml. The sedimentation coefficient was calculated at 62 S. The nucleoprotein consisted of 5 % single stranded RNA and a capsid protein of molecular weight 33.500 daltons. Large quantities of non-capsid proteins were isolated from infected tissue with a molecular weight of 17.500 and 16.500 daltons. Peregrinus maidis, injected with purified MStpV preparation failed to transmit the disease to healthy plants. However, they were infectious when injected with clarified infected plant sap. Antisera against capsid and non-capsid proteins from MStpV-Florida strain reacted positively with the Venezuelan antigens. 相似文献
16.
Jose P. Llongueras Saraswathy Nair Dayana Salas-Leiva Andrea E. Schwarzbach 《Molecular biotechnology》2013,53(3):249-256
A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A 260/A 280 between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A 260/A 280 ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A 260/A 280 measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis. 相似文献
17.
A simple and efficient protocol involving the use of cetyltrimethylammonium bromide (CTAB) for rapid isolation of high molecular wt ( 50 kb) DNA from Streptomyces aureofaciens is described. The DNA yields range from 1.5–2.5 mg per 1.0 g (wet wt) of mycelia, with the purity measured at A260/280 of 1.83–1.97 and also at A260/230 of 2.2–2.71. The DNA preparation is suitable as substrate for restriction digestion, Southern hybridization and library construction. © Rapid Science Ltd. 1998 相似文献
18.
Tancevski I Wehinger A Patsch JR Ritsch A 《International journal of biological macromolecules》2006,39(1-3):77-82
Recombinant adenoviral vectors encoding human HDL-cholesterol receptor SR-BI (Ad/hSR-BI) or beta-galactosidase (Ad/lacZ), respectively, were purified using a Source15 Q anion-exchange (AEX) column and quantified using two parallel Taqman Real Time PCR systems with different target sequences. Adenovirus concentrations were ascertained by 260 nm measurements, purity by 260/280 nm ratio and SDS-PAGE. Subsequently, adenoviruses were validated by experimental intravenous application into New Zealand White rabbits. Transgene expression was verified by functional assays determining plasma clearance rate of 3H-HDL-cholesterol, and was not affected by 21-months storage at -80 degrees C. No alterations of liver enzymes and C-reactive protein (CRP) upon Source15 Q adenovirus treatment could be detected, demonstrating biological safety of our protocol. 相似文献
19.
A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented. 相似文献