磁珠法快速提取基因组DNA的实验研究

针对临床标本基因组DNA的提取方法缺乏广泛适用性,提取步骤繁琐,需要进行离心操作,并且提取过程中会用到苯酚、氯仿等有机试剂,会对操作人员有一定的危害性等不足,本研究拟建立一种适用于临床标本的基因组DNA快速提取方法。在传统的基因组DNA提取试剂和方法的基础上,本研究采用二氧化硅修饰的超顺磁珠设计了一种基因组DNA快速提取方法;探讨磁珠用量、裂解液p H、盐酸胍浓度等因素对基因组DNA提取效率的影响并采用凝胶电泳实验进行验证。当磁珠用量在50~100μg/100 mg样品,裂解液p H约为6,盐酸胍的浓度为6 mol/L时基因组DNA提取效果好、效率高,且磁珠的用量与吸附表面积成正比,但达到一定用量后不会增加提取量,对于100 mg肺组织,适宜的磁珠用量为80μg。此基因组DNA提取方法高效省时,简便快捷,性价比高,适用临床大量样本基因组DNA提取。     

磁珠法在微量检材DNA提取中的应用

目的:研究磁珠法提取微量生物检材DNA物质的可行性。方法:对汗斑、微量唾液斑、指甲这3类微量生物检材采用磁珠法提取DNA,经过STR荧光复合扩增,ABI310测序仪检测,进行STR分型,以检测提取方法的灵敏度。结果:这3类微量生物检材获得了完整的STR分型。结论:磁珠法提取微量检材中DNA物质的效果较好。     

双磁珠法质粒DNA提取技术及应用

质粒是基因合成与测序领域中使用最为频繁的基因运载工具,然而传统的质粒DNA提取方法面临提取通量低、生产成本高等问题,无法满足日益增长的需求。本研究基于质粒提取原理,开发了双磁珠法(double-magnetic-bead method, DMBM)质粒提取技术,探究了磁珠投入量、质粒DNA片段大小、菌液投入量等因素对质粒提取的影响,并且对比了本技术与商业化质粒DNA提取试剂盒提取DNA质量、提取通量及提取成本。结果表明,双磁珠法质粒DNA提取技术可满足不同细胞密度、不同片段长度的质粒DNA提取。此外,该技术搭载96通道全自动核酸提取仪,提取的质粒DNA纯度更高、提取时间缩短80%、提取成本缩减57.1%,从而实现了质粒DNA提取的高通量、低成本,有效助力基因合成与测序。     

浅谈DNA的粗提取和鉴定实验的设计思路

遵循实验步骤提取和鉴定DNA．从而掌握“怎样做”的方法十分重要。但亲身体验科学过程．培养“为什么要这样做”的思维习惯,使学生改变学习方式而主动学习就更为重要。下面就谈一谈该实验的设计思路。     

"DNA粗提取与鉴定"实验的改进

新高中《生物》教材中 ,增加了“DNA的粗提取与鉴定”的实验 ,目的在于 :1 )学会 DNA的粗提取和鉴定方法 ;2 )观察提取出来的 DNA。该实验拓宽了与物理学和化学的联系 ,强调了发现知识的过程与方法 ,减少学生需要记忆的文字内容。同时也强调生物学的科学地位。在 2 0 0 0年 1 2月由人民教育出版社出版的《生物·第二册》(试验修订本·必修 )里 ,要求学生做“DNA的粗提取与鉴定”的实验 ,该实验以新鲜鸡血为原料 ,其提取流程略。而在与之相配套的由人民教育出版社出版的《教师教学用书》里 ,又介绍了另一种“DNA粗提取和鉴定”的方法 ,…     

“DNA的粗提取与鉴定”实验的改进

"DNA的粗提取与鉴定"实验是人教版高中生物学中的一个重要实验,但教材中给出的实验方案存在一些不足。通过多次实验,从提取DNA的实验材料、提取方法、鉴定方法等方面进行了改进,取得了较好的实验效果。     

微波法快速提取放线菌基因组DNA

原有的放线菌基因组DNA提取方法费时、费力、费用高,且对极端环境放线菌成功率较低。利用微波热振惊提取固体培养基表面放线菌菌落基因组DNA,具有快速、简便、费用低廉等优点。所得的基因组DNA可作为PCR反应的模板进行16S rRNA基因有效扩增。这为大量放线菌菌株的快速鉴别和系统分类创造了条件。     

实验动物皮肤病原真菌DNA快速少量提取法

PCR技术应用于实验动物皮肤病原真菌检测,方法简单、省时。但是,真菌的DNA提取较为困难。本文推荐一种既简单又经济快速的提取皮肤真菌DNA的方法,并能成功用于实验动物皮肤病原真菌质量检测研究。     

稗草DNA的快速提取研究

优化了稗草DNA的提取条件,水浴时间为60min,无水乙醇用量为0.60ml,细胞提取液用量为6．0ml,饱和NaAc溶液用量为1.0ml,酚／氯仿／异戊醇溶液、氯仿／异戊醇溶液和异丙醇与样液体积比别为0．60、1．0和0．60。从稗草种子提取时,产率为430μg／g。琼脂糖凝胶电泳证实,所提稗草DNA无断裂降解。     

"DNA的粗提取与鉴定"实验的改进

“DNA的粗提取与鉴定”实验是高中生物学教学中的一个重要实验,此实验教材中是用鸡血作为实验材料,实验时存在实验操作繁琐,学生一堂课很难完成实验。实验过程中较难观察,在析出DNA黏稠物时,难以估计加入蒸馏水的量等问题,实验费用太高,对普通学校来说,买活鸡显然不现实,与市场上的小商贩联系购买新鲜鸡血,很难定时、定量地保证供应。为此我们对此实验加以改进：[第一段]     

A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue

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.     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

FTA-DNA直接提取法在病原真菌分子鉴定中的应用

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。     

一种快速微量提取植物叶片DNA的方法

介绍了一种快速提取微量DNA的方法。该方法简单易行,无需任何特殊设备,所需样品量少。提取的DNA纯度高,D260nm/D280nm在1．9-2．1之间,可满足RAPD、SSR、转基因植株的PCR检测等以PCR扩增为基础的实验需要。     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.     

一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.     

A novel method for the isolation of mycobacterial DNA

Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to −70 °C, then incubated at 65 °C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 × 10⁹) or more cells) including Mycobacterium neoaurum M. fortuitum M. phlei and M. smegmatis . Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis .     

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.     

一种快速提取基因组DNA的方法

采采用氧化硅超顺磁性纳米磁珠和自己设计的试剂体系及提取流程,建立了一种基因组DNA的快速提取方法,该方法以氧化硅磁珠为固相吸附载体,盐酸胍、 -巯基乙醇和SDS为主要裂解吸附试剂。以全血或培养细胞为实验材料进行基因组DNA的提取结果显示用本文建立的方法提取100 L小鼠抗凝血,可得2～3 g基因组DNA, OD260/OD280为1.8 ± 0.05,其纯度可满足后续的酶切和PCR生物操作要求。该方法整个提取过程只需12分钟,不需特殊实验条件同时可省略蛋白酶K的消化过程和离心操作,适用于一般实验室的需求,是一种操作简便、快速高效的提取方法。     

A rapid and efficient method for the isolation of restrictable total DNA from plants of the genusAbelmoschus

A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved using cotyledons of dark-grown seedlings.