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第二节 动植物组织mRNA 提取 一、材料 水稻叶片或小鼠肝组织。 二、设备 研钵，冷冻台式高速离心机，低温冰箱，冷冻真空干燥器，紫外检测仪，电泳仪，电泳槽。 三、试剂 1、无RNA 酶灭菌水：用将高温烘烤的玻璃瓶（180℃ 2小时）装蒸馏水，然后加入0.01%的DEPC（体积/体积），处理过夜后高压灭菌。 2、75%乙醇：用DEPC处理水配制75%乙醇，（用高温灭菌器皿配制），然后装 ...

与rRNA 和tRNA 不同的是，哺乳动物细胞的绝大部分mRNA 在其3'端均有一poly(A)尾，因此可以用oligo(dT)－纤维素亲和层析法从大量的细胞RNA 中分离mRNA dmonds等，1971;At Leder，1972）。在构建cDNA文库时， 必须经上述纯化步骤制备mRNA 模板。 进行Northern杂交或Sl 核酸酶作用图分析时，与总RNA 相比，采用poly(A)+ RN ...

一、用异硫氰酸胍提取 提禽流感病毒的详细步骤，可参考（我提过N次做定量PCR都没问题）： 1.取200ul样品数+阴性对照+阳性对照个1.5ml灭菌eppendorf管。 2.加600ul异硫氰酸胍，然后加入对照和样品，再加200ul氯仿，颠倒混匀。 3.13000rpm离心15min。 4.在第3步离心快结束时，另取同样多eppendorf管，加入400ul -20度预冷的异丙醇。 5.取第3 ...

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RNA干扰是最近发现的一种功能工具。当RNA导入一个细胞时，最终会引起细胞内互补mRNA的降解，从而导致基因功能活性的阻断。 PTGS转录后基因沉默（posttranscriptionalgenesilencing）；一种首先在植物中确定，然后发现在动物中也存在的现象。尽管PTGS最初被描述作一种病毒防御和转位子沉默的内源方法，现在它已经成为一种新的令人激动的研究工具��RNAi干扰。 协同抑制（ ...

从植物组织中提取RNA 是进行植物分子生物学方面研究的必要前提。要进行Northern杂交分析，纯化mRNA 以用于体外翻译或建立cDNA文库，RT-PCR及差示分析等分子生物学研究，都需要高质量的RNA 。因此，从植物组织中提取纯度高、完整性好的RNA 是顺利进行上述研究的关键所在。 从文献报道上看，有许多植物就是由于未能有效地分离纯化其组织中的RNA ，而阻碍了其分子生物学方面研究的进展。一般 ...

A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells. Wu W Hodges E Redelius J Hoog C. Nucleic Acids Res. 2004 Jan 22;32(2):E17. Center for Genomics and Bioinf ...

Primer Extension analysis of total yeast RNA 1. Mix the following in a 1.5 ml eppendorf tube: a. 20-80 micrograms yeast RNA dissolved in H2O (the amount will depend on the level of expression for the ...

How does RNAi work? Genetic and biochemical data indicate a possible two-step mechanism for RNA interference (RNAi): an initiation step and an effector step. a | In the first step input double-strande ...

First strand of cDNA synthsis 1.Add 2 μl of Not I primer-adapter to a sterile 1.5 ml RNAase free tube add 4.5 μl mRNA of molt4 cells (~5.2 ug) add 0.5 μl DEPC-H2O. 2. Heat the mixture at 70℃ ...

RNA interference (RNAi ) by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs) is a powerful method for preventing the expression of a particul ...

The RNA in situ procedure described below is based on the protocol developed by Tautz and Pfeifle (Chromosoma 98 (1989) p81) but adapted to allow the screening of 96 RNA probes on whole-mount Drosophi ...

General Guidelines siRNA targeted sequence is usually 21 nt in length. Avoid regions within 50-100 bp of the start codon and the termination codon Avoid intron regions Avoid stret ...

Chemicals needed:GlucoseL-(+)-Arabinose Sigma Cat # A3256L-RhamnoseSigma cat # R3875Cb (carbenicillin) Sigma cat # 1389DL-p-Chlorophen ...

N.B: Gloves should be worn at all times during preparation of in vitro RNA. All solutions should be RNase-free i.e. made with DEPC water if home-made or bought specifically to use for RNA work. You w ...

Feature The RNase A is free of DNase activity. It is not necessary to heat it before use. Description The Ribonuclease A (RNase A) is an endoribonuclease that specifically degrades single-stranded RNA ...

本文来自于哈佛大学医学院果蝇RNAi 筛选中心的经典实验方法，专门用于果蝇RNAi 实验方法。感谢哈佛大学医学院果蝇RNAi 筛选中心的支持！ A.Primer Designed dsRNA B.Template Selection C.PCR D.In vitro RNA Transcription E.dsRNA Purif ...

siRNA Design RNAi target selection rules : Targeted regions on the cDNA sequence of a targeted gene should be located 50-100 nt downstream of the start codon (ATG). Search for sequence motif AA(N19 ...

RNA i Glossary Dicer - Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNA s. Dicer processes long dsRNA into siRNA of 21-23 nt. Interferon - A small ...

1.NCBIshRNA资源库 http://cgap.nci.nih.gov/RNAi About RNAi on CGAP The NCBI is part of the consortium supporting the preparation of human and mouse libraries containing RNAi constructs that target cance ...