一种新的DNA银染方法

本文介绍一种有效的聚丙烯酰胺中的DNA银染方法,其具有背景浅、快速、灵敏度高等特点。利用该方法对MarkerX和棉花的RAPD产物进行银染,取得了较满意的结果。说明该方法适于聚丙烯酰胺中的DNA银染。 Abstract：An effective silver staining protocol for DNA in PAGE was introduced in this article,it characterized weak background,sensitivity,time saving .Based on this protocol,MarkerX and products of RAPD were stained,the satisfactory staining map indicated that this silver staining protocol was applicable to DNA in PAGE.     

一种简单快速高分辨率的PAGE胶显带方法

尽管使用常规方法对聚丙烯酰胺胶(PAGE胶)进行DNA显带, 能获得高分辨率图片, 但常规方法操作步骤繁琐, 耗时较长。文章报道了一种PAGE胶DNA显带的改进方法, 分别使用改进的显带方法和常规的显带方法进行了PAGE胶的显影和比较。结果表明, 使用改进的显带方法得到的PAGE胶图片, DNA带型和背景具有更高的对比度, 因此具有更高的分辨率; 同时操作步骤少, 耗时短, 使用试剂少。这种方法在本实验室中已经完全代替了常规PAGE胶显带方法。     

脂多糖快速银染检测方法的改良

为了建立一种快速、高效的脂多糖(LPS)银染检测方法,利用大肠杆菌(Escherichia coli 0111:B4)制备的LPS为研究对象,在传统LPS银染检测方法的基础上,通过调整凝胶的固定氧化试剂和银染试剂等措施,对LPS经SDS-PAGE电泳后的结果进行银染检测.结果表明,改良的LPS快速银染检测方法在保持传统方法灵敏度的基础上,操作过程更为简单,安全性、经济性和染色效果等方面也比传统的方法均有明显提高,是一种快速高效的LPS银染检测方法.     

微卫星PCR产物变性与非变性PAGE-银染检测方法的比较

用鸡的微卫星引物对6个中国地方鸡种的两个微卫星位点进行PCR扩增。将扩增产物在变性与非变性聚丙烯酰胺凝胶（PAGE）上进行电泳,经银染,表明微卫星产物在二者上的电泳结果有明显的差异。在非变性聚丙烯酰胺凝胶中,表现为有较多的非特异带,而在变性胶中微卫星扩增产物条带清晰,易于鉴定。Abstract：The genomic DNAs from six chicken breeds in China were amplified using two microsatellite primers. The PCR products were detected by non-denatured and denatured PAGE gels respectively, and the gels were dyed by silver. There were distinct differences between the two kinds of gel. In non-denatured gels. There were many nonspecific bands while clear purposed bands were showed in denatured gels.     

氯化钇和氯化镨引起的人淋巴细胞DNA分子损伤的研究 A Study of DNA Damage in Human Lymphocytes Induced by Yttrium Chloride and Praseodymium Chloride

用单细胞凝胶电泳法检测了稀土化合物氯化钇和氯化镨对人外周血淋巴细胞的DNA损伤效应。结果表明,与对照相比,3种不同浓度的氯化钇和氯化镨均可引起淋巴细胞DNA受损后DNA迁移率的显著升高,受损伤细胞的百分率与对照差异明显,提示氯化钇和氯化镨具有一定的遗传毒性。 Abstract:The effect of DNA damage in human lymphocytes induced by yttrium chloride and praseodymium chloride was detected using SCG assay.The results showed that a highly significant increase in DNA migration in DNA-damaged lymphocytes was induced by three different concentrations of yttrium and praseodymium compared with the control,The percentage of DNA-damaged have genetic toxicity.The relevant points to this study are discussed.     

一种简单高效的食用真菌总RNA提取方法

以金针菇为材料,建立了一种适合于富含RNase、多酚、多糖和糖蛋白的食用真菌RNA的提取方法,此方法在高浓度变性剂存在的条件下2次用苯酚-氯仿-异戊醇进行抽提去除DNA、蛋白质,并用异丙醇和乙酸钠选择性沉淀RNA、去除多糖,得到完整、均一的RNA样品。 Abstract:With Flammulina velutipes material,an improved method was developed for extracting total RNA from domestic fungus that are rich in RNase,polyphenols,polymeric carbohydrates and proteoglycans..Phenol-chloroform-isoamyl alcohol were used twice to clear DNA and protein under higher concentration of denaturing solution and isopentanol,sodium acetate were used to precipitate RNA selectively.Pure and intact RNA can be effectively prepared by this method.     

一种新的DNA芯片的封闭方法

提出了一种新的DNA芯片的封闭方法：1-碘丙烷封闭方法,并对1-碘丙烷和琥珀酸酐两种不同的封闭方法的封闭效果进行了比较实验,研究封闭试剂、封闭时间对氨基DNA芯片杂交结果所产生的影响。结果表明： 1-碘丙烷封闭效果明显好于琥珀酸酐的封闭效果,用1-碘丙烷封闭后的芯片进行杂交,芯片杂交信号强、背景噪声低,灵敏度高,整个封闭过程实验操作简单,时间短,1h即可达到所需效果。1-碘丙烷可以作为一种很好的DNA芯片封闭试剂。Abstract: Suggested a new blocking method for DNA microarray: 1- iodo -propane .we compared the difference between the two block reagents and the blocking results in different blocking time. The results showed that 1-I-prppane is batter-blocking reagent than succinic anhydride in DNA chip.1h blocking time is enough for blocking.     

保存方法和保存时间对竹子核DNA含量 （2C-值） 检测的影响

竹子核DNA含量（2C 值）的检测对竹资源的科学研究具有重要意义,而大部分野外采集的样本,通过不同方法保存后,均使用流式细胞仪技术进行核DNA含量检测。本文选取麻竹（Dendrocalamus latiflorus）、筇竹（Chimonobambusa tumidissinoda）和毛花酸竹（Acidosasa purpurea）三种竹子样本,使用硅胶保存法和Sample protector试剂保存法分别保存4d、8d、12d、16d后,采用流式细胞术检测样品2C 值。CV值可以用来反映数据检测结果质量,是对检测结果准确性以及精确性的评价标准,我们通过样品CV值的大小和2C 值的变异率来评价保存方法和保存时间对竹子2C 值检测的影响。方差分析显示,保存时间对CV值具有显著性影响（P < 0001）,随着保存时间增大,CV值增大;保存方法对2C 值变异率有显著性影响(P < 0001)。硅胶保存法保存后的样品,测量值比新鲜材料大;Sample protector试剂保存法保存后,测量值比新鲜材料小。因此,随着保存时间的增加,样品CV值增大,引起检测结果质量降低。研究发现,硅胶保存法和Sample protector试剂保存法会影响竹子样本2C 值大小,但2C 值大小的变化小于10%。     

介绍一种简单高效的植物总RNA提取方法

在液氮中研磨小麦幼叶和不同发育时期的种子,经含0.1% SDS和0.1%十二烷基肌氨酸钠(LDS)的尿素缓冲液裂解后,醋酸钠和氯仿沉淀变性蛋白质,异丙醇沉淀核酸,溶解后经2.5mol/L LiCl沉淀总RNA,洗涤后就可得到高质量的总RNA,其OD260/OD280 为2.05~2.10,28S和18S RNA带清晰,叶片总RNA还可得到23S和16S RNA带,产率可达5mg RNA/10g材料。当使用含1% SDS和1% LDS的尿素缓冲液裂解材料时,则可用于DNA的分离提取,其分子大小可达50~100kb以上。 Abstract:Wheat leaf and seeds at different development stages had been squashed in liquid nitrogen,then lysised by urea buffer which contains 0.1% SDS and 0.1% LDS,denatured protein had been removed by NaAc and chloroform precipitation,total RNA was further purified by LiCl.The RNA we obtained had sharp bands of 28S and 18S after agarose gel electrophoresis,23S and 16S RNA bands can also be seen clearly in leaf RNA extract,the value of OD260/OD280 of RNA was 205~210.5mg RNA can been isolated from 10g leaf of wheat.This method can also been used in high molecular weight DNA isolation but the concentration of SDS and LDS must be increased to 1%.     

保存方法和保存时间对竹子核DNA含量（2C-值）检测的影响

竹子核DNA含量（2C-值）的检测对竹资源的科学研究具有重要意义,而大部分野外采集的样本,通过不同方法保存后,均使用流式细胞仪技术进行核DNA含量检测。本文选取麻竹（Dendrocalamuslati—florus）、筇竹（Chimonobambusatumidissinoda）和毛花酸竹（Acidosasapurpurea）三种竹子样本,使用硅胶保存法和Sampleprotector试剂保存法分别保存4d、8d、12d、16d后,采用流式细胞术检测样品2c．值。CV值可以用来反映数据检测结果质量,是对检测结果准确性以及精确性的评价标准,我们通过样品CV值的大小和2c-值的变异率来评价保存方法和保存时间对竹子2c-值检测的影响。方差分析显示,保存时间对CV值具有显著性影响（P〈0．001）,随着保存时间增大,CV值增大;保存方法对2c-值变异率有显著性影响（P〈0．001）。硅胶保存法保存后的样品,测量值比新鲜材料大;Sampleprotector试剂保存法保存后,测量值比新鲜材料小。因此,随着保存时间的增加,样品CV值增大,引起检测结果质量降低。研究发现,硅胶保存法和Sampleprotector试剂保存法会影响竹子样本2c．值大小,但2c-值大小的变化小于10％。     

微卫星DNA聚丙烯酰胺凝胶电泳(PAGE)银染法的改良

为了筛选和检测绵羊种群中具有较好表现的微卫星多态性,采用Touch-down PCR方法扩增绵羊基因组上的微卫星DNA序列后,经聚丙烯酰胺凝胶电泳分离,采用改良的银染法对微卫星位点进行多态性检测,染色结果与常规银染法相比,该方法具有灵敏度高、背景浅、污染小、条带清晰等突出特点,能有效地控制染色背景,显著提高检测分辨率,整个染色过程所需时间短,具有广泛的推广价值。     

纳米金标记-逐步银染法快速检测金黄色葡萄球菌

将纳米金探针应用于目的核酸的检测,具有与PCR相当的灵敏度和特异性.本研究建立了一种可以在微孔板上快速检测金黄色葡萄球菌的纳米金标记-逐步银染法.该方法利用已包被链霉亲和素的微孔板,将PCR扩增的金黄色葡萄球菌nuc基因与生物素探针、纳米金探针形成的三明治杂交结构锚定其上,然后在低温下逐步银染显色,通过酶标仪检测放大的银染信号.这种纳米金标记-逐步银染法可以在显著降低非特异性背景信号的同时放大银染信号,检测金黄色葡萄球菌nuc基因的灵敏度为1 pmol/L,比常温一步银染法的灵敏度提高约102倍. 51例临床标本的检测结果与PCR法一致,与培养生化鉴定法的检测结果之间无显著性差异(P >0.05). 综上所述,本研究成功构建了金黄色葡萄球菌的纳米金标记-逐步银染法,在病原微生物的快速检测领域表现出广阔的发展潜力.     

A Rapid Silver Staining and Destaining Technique for the Nucleolus Organizer Region

Silver staining of nucleolar organizing regions (NOR) is common, but a standard protocol is lacking. A modification of a rapid silver nitrate staining technique for NORs is presented here. Advantages of the modified technique include reliability, speed, cost and the fact that it can be carried out in the light.     

银染增强的纳米金标记探针对微量核酸的检测

本研究利用银染增强的纳米金技术建立了一种简单快速的核酸定量方法.该方法基于纳米金与烷巯基修饰的寡核苷酸分子共价键合作用,将纳米微粒报告基团标记在与靶核酸一端序列互补的寡核苷酸上,同时生物素化修饰另一端互补序列.靶核酸与两段寡核苷酸探针杂交后,借亲和素固定在酶标板孔内,通过纳米金催化的银染放大效应产生高灵敏的识别信号,适时记录其吸光度值从而实现核酸分子的定量.该检测方法检测单链核酸分子的灵敏度达0.1 fM,双链分子为10 fM.     

A Rapid and Simple Method for Staining Lipid in Fixed Nematodes

A method is described for staining lipid in fourth-stage dispersal juvenile nematodes fixed with formal-acetic fixative (FA4:1). Bursaphelenchus xylophilus fourth-stage dispersal juveniles were fixed with hot FA4:1 for 24 hours, excess fixative was removed, and a solution of saturated oil red O in 96% ethanol added and allowed to sit for 25 minutes at 60 C. Excess oil red O was removed, nematodes were washed twice with 70% ethanol, and were processed to pure glycerin. Lipid droplets within the nematodes were viewed by light microscopy and appeared as dark red spheres of various sizes. Computerized image analysis was used to quantify lipid droplet area.     

乳酸脱氢酶与酯酶同工酶同板染色法

介绍一种在同一块凝胶板上染乳酸脱氢酶(LDH)与酯酶(EST)的染色方法. 该同板染色法利用两种同工酶显色反应互不干扰和颜色不同的特点, 先染LDH, 后染EST, 可以在同一块胶板上得到两种同工酶清晰的酶带, 每一种酶的酶带与单板染色的酶带完全一样. 这种染色法, 能节省同工酶分析所需的试剂、时间和经费, 也便于样品的鉴定与比较, 是一种经济有效的方法. 此方法, 同样适用于苹果酸脱氢酶(MDH)与酯酶等同工酶的同板染色.     

用SDS-PAGE和Vorum银染法比较真菌菌丝体破壁效果

目的:研究不同处理方法对丝状真菌Pythium sp.GY1938菌丝体破壁效果的差异。方法:采用7种方法对真菌菌丝体进行破壁处理,将制备的胞内可溶蛋白质样品通过SDS-PAGE和Vorum法银染处理,比较不同破壁方法处理后蛋白质释放效果的优劣。结果:方法VI(石英砂、液氮、研磨)的破壁效果最好,蛋白质的分离效率和提取质量最高。结论:方法VI适合于丝状真菌Pythium sp.GY1938蛋白组学研究中目的蛋白的提取。     

A Rapid Safranin-Metal Phthalocyanine Double Staining Technique for Plants

Pure metal 4.4',4',4'-tetxa-substituted, sulfo-, carboxy- and nitrophthalocyanines were synthesized. Mounted, deparaffinized and partially dehydrated sections of plant tissues were stained with 0.5% safranin in 50% alcohol for 5-10 min. Excess safranin was removed with a series of 70%, 95% and absolute alcohol washes. The sections were then stained for 2-3 min using metal 4,4',4',4'-phthalocyanine tetracarboxylic acid (MPTC, 0.5% (V/V) containing a few drops of dilute sodium hydroxide), metal 4,4',4',4'-tetra-sulfophthalocyanine (MPTS, 0.5% (V/V)) or metal tetranitrophthalocyanine (MPTN, 0.5% (V/V) in dimethyl sulfoxide). The sections were washed with 95%, then absolute alcohol; however, the metal tetranitrophthalocyanine section was washed only with absolute alcohol. Stained sections were treated briefly with xylene, then mounted on a coverslip. Bright peacock blue (MPTC and MPTS using Cu, Co or Ni), turquoise blue (MPTN using Cu or Ni) or parrot green (zinc phthalocyanine tetracarboxylic acid-ZnPTC, zinc phthalocyanine tetranitro derivative-ZnPTN) colors were obtained. Lignin-containing cells were stained red by safranin and the remaining cell structures were stained by the metal phthalocyanine complex with color brightness superior to that of fast green. Uniform staining, no color fading after a year, reliability, brief staining times, high color contrast (log ε = 4.0-4.9) and ease of use make this double staining combination ideal for routine use and photomicrography.