风沙土微生物总DNA不同提取和纯化方法比较

为筛选和建立风沙土中总DNA的提取和纯化方法,选取了5种直接提取法、1种间接提取法和2种纯化法分别对风沙土中总DNA进行了提取和纯化,并对其质量和产量进行了比较.结果表明：6种方法均可从风沙土中提取到大小为23 kb左右的总DNA,其中改进后的高盐提取法（用40%聚乙二醇8000和4 mol·L^-1 NaCl沉淀DNA）效果最好,纯化后总DNA的纯度最高,可进行16S rDNA的PCR扩增,且产量仅稍低于试剂盒提取法;电泳加柱回收纯化法的纯化效果较好,经该方法纯化后的总DNA大部分可进行PCR扩增,可满足后续分子操作对DNA纯度的要求.     

八角莽草酸不同纯化方法的比较研究

利用重结晶、醇沉和柱层析等方法对八角莽草酸进行提取纯化,并比较了不同方法的纯化结果。结果表明:乙醇沉淀法的纯化效果明显,醇沉后进行硅胶柱或大孔树脂柱层析分离纯化莽草酸的效果都大为提高,纯度都达到82%以上。利用一种新的洗脱体系-乙酸乙酯和甲醇的混合溶剂,用于硅胶柱层析法纯化莽草酸,能将莽草酸与其近似物很好地分离,纯度达到94.6%,纯化的效果优于大孔树脂柱。最终确定了高效提取纯化莽草酸的工艺流程:微波提取——醇沉法初步纯化——柱层析法进一步纯化。     

接触DNA常规提取纯化方法的比较探讨

接触DNA的提取纯化是法医物证工作的重点和难点之一。接触性检材上的脱落细胞本就量小体微,所以运用有效的方法对这些潜在的微量脱落细胞中的DNA进行提取纯化就显得十分重要。文章拟对接触DNA的常规提取纯化方法及影响因素进行简要阐述,并对其进行横向的比较探讨,以期得出最有效的接触DNA提取纯化方法。     

蛹虫草中虫草素的提取及纯化工艺研究

目的:优化从蛹虫草中提取、纯化虫草素的方法并鉴定其纯度.方法和结果:通过3种提取方法的比较,确定了以恒温水浴法作为虫草素规模化提取方法,最佳提取条件为45℃、80％乙醇;同时,确定了ML-7大孔树脂纯化虫草素的工艺,采用该工艺虫草素纯度可达到92％以上,并对纯化虫草素和标准品进行了比较鉴定.结论:该方法提取、纯化的虫草素纯度高,适于虫草素的规模化生产.     

狭叶坡垒基因组总DNA的提取和纯化方法研究

以狭叶坡垒叶片为材料,对基因组总DNA的提取和纯化方法进行了研究,并对改良CTAB法、高盐低pH法和改良SDS法进行比较.结果表明,改良的CTAB法更适合狭叶坡垒基因组总DNA的提取,且硅胶干燥30 d的叶样和新鲜叶样所得的DNA几乎没有区别.再对改良CTAB法的水浴时间进行探索,发现150 min是较为合适的水浴时间.对比酚纯化法和试剂盒纯化法,发现试剂盒纯化损失的DNA少,得率高,是一种简便、快速、安全的纯化方法.     

花青素提取,分离及纯化方法的研究现状和发展趋势

本文就花青素的结构和种类进行了简单介绍,并且对花青素提取、分离及纯化的方法进行了详细综述,对其未来的发展趋势进行了合理展望,希望对花青素提取分离工作有一定的参考意义。     

土壤微生物总DNA的提取和纯化

本文建立了从土壤中提取总DNA的方法,并通过改进使适合于对革兰氏阳性菌的提取。用9种性质不同的土壤进行验证,均提取到了DNA,每克干土的DNA提取量从3.30μg～13.41μg,通过透析袋回收进行纯化,纯化回收率达到65.34%,纯化后的土壤DNA可以直接扩增出16S rDNA。9种土壤的提取率从60.51%～93.45%,可以从每g干土添加362个菌体的土壤中扩增到目的条带。     

巨尾桉叶中皂苷的提取分离及其抗氧化活性研究

开展了从巨尾桉叶中提取皂苷的工艺优化研究.考察了温度、时间,溶剂用量对皂苷得率的影响,并对粗提物进行纯化.结果表明,巨尾桉叶皂苷的优化条件是:提取温度61.8℃,提取时间5.02h,溶剂与巨尾桉叶的液料比为20.09∶1,皂苷的提取得率(g/g)为5.51％.提取物经大孔树脂-丙酮沉淀联用分离法纯化后皂苷含量由16.22％提高到50.35％.对粗提物、大孔树脂纯化物和大孔树脂吸附-丙酮沉淀纯化物进行抗氧化活性研究.结果表明,它们对DPPH自由基的最大清除率分别为83.02％、84.20％和85.62％,对过氧化氢的最大清除率分别为80.10％、55.02％和48.21％,对超氧阴离子自由基的最大清除率分别为16.62％、13.43％和10.01％.     

树脂法提取纯化白藜芦醇的新工艺研究

对虎杖中的白藜芦醇先酶解再提取,提取液采用一种新树脂进行纯化分离。考察了酶解物、不同树脂、上柱量、洗脱量等因素对白藜芦醇提取纯化的影响,确立了白藜芦醇的最佳提取纯化条件:用酿酒曲于35~40℃将原药材先酶解24 h,再用75%乙醇提取,提取液经回收乙醇后上XC-7大孔树脂柱,先加10%乙醇洗脱,然后再用60%乙醇洗脱并收集洗脱液,浓缩,真空干燥即可,产品中白藜芦醇含量达85%,白藜芦醇转化率达85%以上。     

酵母源葡萄糖耐量因子的提取纯化研究

为探究葡萄糖耐量因子(GTF)分离纯化工艺,简化纯化步骤,以期得到GTF纯品,本实验在前人氨水提取基础上,采用有机溶剂沉淀除去杂蛋白,在有机溶剂的种类、溶剂的浓度、提取组分以及沉淀时间的确定等方面进行了试验,以有机铬含量(μg)与总蛋白含量(mg)的比值为纯化评价标准,高相液相色谱对纯化结果进行验证,同时将纯化前后的GTF作用于胰岛素抵抗型Hep-G2肝癌细胞,测定细胞葡萄糖消耗量,检测GTF纯化品对细胞葡萄糖代谢的调节活性。最终确定用30%低温乙醇沉淀20 min,10000×g离心取上清液分离纯化GTF,此方法相较于氨水直接提取,有机铬含量与总蛋白含量的比值提高了6倍多,GTF提取率和纯度都得到了显著提高,且纯化后的GTF对胰岛素抵抗型Hep-G2细胞葡萄糖代谢具有显著调节作用(P0.01)。     

Large-scale purification of oligonucleotides by extraction and precipitation with butanole

Today the synthesis of oligonucleotides is a well-established process. Using automatic synthesizers even kilogram quantities can be produced in a few hours. However, the purification of the final product is still time-consuming and needs a complex apparatus. In this article, a simple and fast purification method for the large-scale syntheses of oligonucleotides is described. According to the method of Sawadago and van Dyke ([1991] Nucleic Acids Res 19:674-675) for small-scale oligonucleotide purification, oligonucleotides in mumol to mmol amounts were purified by liquid-liquid extraction using butanole as the extraction liquid. Choosing appropriate ratios of extraction liquid to oligonucleotide solution, simultaneous purification and precipitation could be achieved. It was found that the yield of the purified oligonucleotide was mainly affected by the temperature. Yield decreased with increasing temperature. The use of this improved extraction procedure allows the purification of gram to kilogram quantities of oligonucleotides in less than a day with simple equipment and high yield.     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

14型肺炎链球菌荚膜多糖纯化工艺中两种去除蛋白方法的比较

目的苯酚抽提法和脱氧胆酸钠沉淀法去除14型肺炎链球菌荚膜多糖中蛋白质的效果比较。方法将3批次14型肺炎链球菌发酵培养液经超滤、乙醇沉淀等方法初步纯化后,平分成两份,分别采用苯酚抽提法和脱氧胆酸钠沉淀法去除蛋白,通过比较多糖收获量、多糖组分检定结果、多糖分子质量、多糖抗原活性、多糖核磁共振图谱,以此评价这两种蛋白去除方法的效果。结果与苯酚抽提法相比,脱氧胆酸钠沉淀法制备的14型肺炎链球菌纯化荚膜多糖除收获量较高,蛋白和核酸杂质含量较低外,氨基己糖含量、多糖分子质量、抗原活性和多糖核磁共振图谱的检定分析结果无显著性差异(P>0.1)。结论作为14型肺炎链球菌荚膜多糖纯化工艺中的除蛋白方法,脱氧胆酸钠沉淀法优于苯酚抽提法。     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Purification of native dehydrin from Glycine Max cv., Pisum sativum,and Rosmarinum officinalis by affinity chromatography

An improved method for the purification of dehydrin from soy (glycine max) is described. Acidic extraction of soy whey was followed by a three step chromatographic process: capture on copper charged Chelating Sepharose Big Beads, intermediate hydrophobic interaction chromatography on Source 15 PHE, and a polishing step on blue Sepharose. The 32-kDa native soy dehydrin was purified to a purity of greater than 98.5% with an overall recovery of 63%. When compared to a previously published purification procedure, recovery, time requirements, and sample preparation steps were improved. The developed method is readily scaleable. Preliminary results show that the process can be used for dehydrins from rosemary (Rosmarinum officinalis) and pea (Pisum sativum).     

Direct process integration of extraction and expanded bed adsorption in the recovery of crocetin derivatives from Fructus Gardenia

A process that integrated an extraction tank (EXT) and an expanded bed adsorption (EBA) into a new system EXT-EBA for direct purifying crocetin derivatives from Fructus Gardenia was described. Conditions were set to allow the extraction and purification in a single step. A comparison between the integrated process and the conventional process to purify crocetin derivatives was presented. The integrated process resulted in 52.79% recovery of crocin compared to 24.12% in the conventional process. The process time and solvent used were decreased in the integrated process. The result suggests that the EXT-EBA integrates extraction, clarification, and purification in a single step, greatly simplifying the process flow and reducing the cost and time of extraction and purification of crocetin derivatives from Fructus Gardenia.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Application of high-performance capillary electrophoresis to the purification process of Escherichia coli K4 polysaccharide

The high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) technique was applied to the extraction and purification process of the K4 polysaccharide from cultured bacteria in several stages. HPCE proved to be a technique with high resolution and sensitivity in analyzing K4 polysaccharide during its purification, in particular by using a strong anion-exchange resin. This is of paramount importance to monitor the product during the extraction and purification process or to test the purity of the final product. Furthermore, HPCE is able to verify that the extraction and purification process adopted is not carried out under drastic conditions capable of inducing fructose removal from the polysaccharide backbone.     

Optimization of micellar extraction for the pre-purification of paclitaxel from<Emphasis Type="Italic">Taxus chinensis</Emphasis>

Currently, there are many reports in the literature regarding technological methods for paclitaxel purification. However, there have been few reports on the purification of paclitaxel using a micellar system. This method is based on the transfer of paclitaxel within the crude extract to an aqueous surfactant solution as a micelle, allowing the use of organic solvents to be used for the removal of lipids and non-polar impurities. In this study, we optimized the important process parameters of micellar extraction to obtain a high purity and yield of paclitaxel in a pre-purification step. The optimal surfactant (N-cetylpyridinium chloride, CPC) concentration, initial crude paclitaxel concentration, organic solvents (methylt-butyl ether/hexane) ratio, extraction temperature, and extraction time were 7.5% (w/v), 16.4 mg/mL, 1.5/1 (v/v), 25°C, and 30 min, respectively. The crude extracts from the liquid-liquid extracts were efficiently pre-purified by micellar extraction, increasing in purity from 6% to over 21%, with a yield of 92%. Overall, the use of micellar extraction in the pre-purification process allowed for rapid and efficient separation of paclitaxel from interfering compounds, and dramatically increased the yield and purity of the crucle paclitael for subsequent purification steps.     

核酸提取方法进展

核酸提取是分子生物学的基本方法,也是核酸诊断中最关键的方法,它是下游诊断、分析和制备的前提。过去的核酸提取方法繁琐费时且有限。目前,核酸提取方法已经取得了较大进步。本文综述了核酸提取方法的进展情况,包括传统的基于溶液的抽提方法、现在常用的柱提取法、正兴起的多效生物分子抽提法和自动化抽提系统等。多效生物分子抽提法、自动抽提系统的微型化和完全自动化或者它们的组合是核酸提取技术未来发展的方向。