介绍一种简便快速的电洗脱方法

凝胶电泳是研究核酸、蛋白质等生物大分子的一项重要技术,也是 DNA 的限制性内切酶切割片断的分析、分离、纯化的一种不可缺少的方法。这样,就时常需要将某些大分子或片段从凝胶中分离纯化出来,所采用的方法很多,如渗滤法、连续电泳洗脱法、透析法等等。但是,所有这些方法都或多或少地受到诸如分子大小、回收率、纯化时间等方面的限制。联邦德国 Schleicher and Schuell 公司最近推出了新型的电洗脱仪,用于从凝胶中分离生物大分子,并可对样品进行浓缩。经我们实验室使用,效果很好。现特作介绍,供国内同     

介绍一种简便的RNA分离制备方法

用酸性异硫氰酸胍-苯酚-氯仿混合物,从人外周血白细胞、组织培养的细胞株及人胚组织中分离制备总RNA,所得产品纯度高,未降解,方法简便,可以同时操作较多的样品。此法确是一种较好的RNA分离制备方法,值得推广。     

一种简便快速检测质粒DNA的方法

简便、快速检测质粒DNA的方法,无论是 在筛选、鉴定新质粒或重组质粒的研究中都具有重要的意义。我们在将质粒pBR 322 DNA与 A DNA进行体外重组、筛选和鉴定重组子过程 中,摸索了一种简便快速检测质粒及重组质粒 DNA的方法。此法对菌体和溶菌产物不需任 何处理,比较简便易行     

一种简便快速筛选重组子的方法

目的：根据碱裂解法抽提质粒DNA的原理,研究应用快检缓冲液方法挑取重组子克隆,从而获得简单易行,快速方便,节省时间,提高工作效率的筛选重组子的方法。方法：不需抽提质粒,只要将菌落接入快检缓冲液后直接进行普通琼脂凝胶电泳分析,就可以快速筛选出重组子。结果：结果和提取质粒酶切鉴定结果一致。结论：经实验证明用快检缓冲液方法筛选重组子是一种简单易行,快速方便,节省时间,提高工作效率并且可靠的方法。     

一种快速简便的噬菌体DNA的抽提方法

噬菌体DNA的制备和纯化是分子生物学研究中极为重要的一环,在基因库和eDNA库的建立,以及DNA克隆和筛选等方面都是不可缺少的。噬菌体DNA的制备可以通过噬菌体感染细菌在琼脂板上繁殖(称为平板法),也可以在液体培养液中繁殖(称为液体法)。平板法多用     

一种简便、快速清洗硅藻细胞壁的方法

硅藻细胞壁的形态结构特征是硅藻分类学的主要依据。       

一种简便,快速的真菌油脂提取方法

利用微生物发酵生产不饱和脂肪酸(ＰＵＦＡｓ)即将大规模工业化生产,诱变筛选富含ＰＵＦＡｓ的高产菌株成为工业育种的热门课题,快速判断真菌中油脂含量对菌株的筛选有着重要意义。对真菌进行油脂含量鉴定的方法有两种:一种是半定量方法———脂肪染色法,该法判断结果较困...     

Improvement of a Simple Method to Purify Ribonucleotide Reductase

Abstract

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported.¹ The column selectively retains < 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KC1 for elution. The enzyme could also be recovered Intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KC1 prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

一种简便的肌醇磷脂微量分析方法

分析磷脂酰肌醇循环(PI cycle)的磷脂组分常采用双向薄层层析法．建立了一个简单快速的单向薄层层析分离肌醇磷脂方法．首先采用不同的有机溶剂体系分别提取非多磷酸肌醇磷脂和多磷酸肌醇磷脂,然后用不同的层析展开体系,对两部分磷脂进行单向薄层层析分离．采用无载体 ³²P标记实验对该方法分离效果进行了观察．此法适用于同位素标记和非标记样品中肌醇磷脂组分的比较分析及多磷酸肌醇磷脂的提取、纯化和定量．     

一种简单、快速提取DNA的方法

随着分子生物学研究的迅速发展,提取ＤＮＡ已成为一项常规实验。用经典方法提取ＤＮＡ［１］,先用蛋白酶Ｋ、ＳＤＳ消化,然后用酚、氯仿抽提,乙醇沉淀,耗时较多,提取液需要多次转移,易引起交叉污染和ＤＮＡ丢失。本文利用硅藻（ｄｉａｔｏｍ）能够特异性吸附核酸的...     

一种简单快速植物组织冰冻切片方法

比较不同冷冻方法对植物细胞超微结构的影响,结果表明:直接包埋法处理的植物细胞超微结构保存较好,而液氮冷冻处理的植物细胞内膜系统损伤严重.建立了一种直接包埋冷冻和适当回温相结合的方法,不仅可以制作出植物细胞基本结构保存完整的组织切片,而且避免了使用冰冻保护剂的弊端.其操作程序是:样品固定→冰冻与包埋→适当回温→快速切片→展片→染色.此法制作的切片可进行不同的染色和组织细胞化学测定,具有操作简便,易于推广的特点.     

A Rapid and Simple Method for Assaying Interferon1

A new procedure of assaying interferon (IF) has been developed. Cell suspension was dispensed into liquid scintillation counting vials together with IF sample. During an overnight incubation, the cells adhered sufficiently to the bottom of the vials and all the subsequent procedures were carried out without transfer of the cells from the vials. Vesicular stomatitis virus was inoculated and virus-specific RNA was labeled by adding ³H-uridine and actinomycin D to the medium. Incubation was terminated prior to completion of a single-step growth of the virus and radioactivity of the labeled cells in each vial was determined. The reciprocal of the IF dilution which reduced the radioactivity in viral RNA by 50% was taken as the titer. The present procedure consists of simple manipulations and can be completed within 24 hr. Furthermore, it is quite reproducible and gives a titer almost identical to that obtained by the conventional plaque-reduction dose method. The procedure can be applied to mouse L cells, rabbit RK-13 cells and human FL cells, without modification.     

一种简单高效的芽胞纯化方法及其效果评价

针对在使用试管扩散法检测食品中抗生素残留总量时出现的检测管制备时间长和保质期短的问题,介绍了一种简单高效的芽胞纯化方法.以嗜热脂肪地芽胞杆菌(Geobacillus stearothermophilus CICC 10392)为例,应用此方法对其芽胞进行纯化后,通过镜检等方式从纯化后芽胞悬液的纯度和保藏2a后芽胞悬液的...     

一种简便快速的聚合酶活性实时检测新方法

基于双链DNA结合染料能特异嵌入双链DNA发出荧光的原理,发展了一种实时检测DNA聚合酶活性的简便方法.在检测过程中,聚合酶的聚合反应进程被实时转换为荧光信号,通过监测荧光强度的变化实时检测聚合酶的活性及药物对聚合酶活性的影响.该方法不需要对DNA进行放射性同位素标记和荧光标记,也不需要聚丙烯酰胺凝胶电泳和聚合酶链式反应,是一种简便、快速的聚合酶活性实时检测新方法,为研究抗肿瘤药物对聚合酶活性的影响提供了一种简捷方法,也将为相关疾病诊治和药物筛选提供一种新的思路.     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

一种简便快速的单引物PCR定点突变方法

为了解决在一些特殊位点上利用Quick Change方法进行定点突变时会在突变位点处额外插入引物序列导致突变失败的问题,对Quick Change法进行了改良。改良方法为:合成在突变位点处点突变的一对反向互补引物,分别进行单引物PCR扩增,将两种扩增产物混合,变性复性后加入Dpn I进行酶切,酶切产物转化大肠杆菌DH5α,抗性筛选阳性克隆进行测序验证。利用此法成功突变紫穗槐二烯合酶(amorpha-4,11-diene synthase,ADS)基因中多个利用常规方法突变均因引入额外引物而无法成功的特殊位点,证明此方法实践上可行,而且也可以避免插入额外引物序列,这也从侧面证明额外引物插入的原因是双引物同时反应。