Trizol说明书

trizol 中文说明书trizol 试剂是一种从组织及细胞中提取总rna的专用试剂。

这种试剂是一种酚和硫氰酸胍的单相溶液，是将chomczynski 和sacchi发明的一步rna提取法的改进。

在样品匀浆或分析过程中，trizol 试剂可在分裂细胞及溶解胞膜的同时保持rna的完整。

在匀浆后加入氯仿，可将溶液分为水相和有机相。

rna均在水相中。

吸取水相加入异丙醇，得到rna沉淀。

去除水相后，样品中的dna及蛋白质可通过进一步连续的沉淀而得到。

中间相加入乙醇可沉淀dna而有机相加入异丙醇则能沉淀出蛋白质。

dna的再纯化可能对标化样品的rna产量有作用。

2．这一技术可以用于人，动物，植物或细菌株的微量组织（50—100mg）及细胞（5×106），也可以用于大量检材（≥1g）及细胞(﹥107).该方法简便易行,可用于同时检测大量样品,全部过程1小时内可以完成.用trizol 试剂分离的rna 没有dna及蛋白质成分.这种rna可用于northern印迹分析,斑点杂交,多聚(a)+检出及vitro转移, rna酶蛋白分析及分子克隆.用于pcr反应时,当两个引物位于同一外显子时应用扩增特异dna酶i处理提取出的rna.3． trizol 试剂可用于分子量从大到小的各种rna的提取.例如,大鼠肝脏提取出的rna,经琼脂糖凝胶电泳,eb染色后可显现出7kb和15kb的大分子量的rna谱带(包括mrna和hnrna), 位于～5kb(28s)和～2kb (18s)的两条主要的核糖体rna谱带及大小在0.1kb-0.3kb之间的低分子量rna(trna,5s).分离出的rna当溶于双蒸水中时a260/280为1.6-1.8之间.每毫克组织所能提取的rna量约为:肝和脾,6-10μg;肾,3-4μg;骨骼肌和脑,1-5μg;胎盘,1-4μg.从1×106培养细胞中提取的rna量约为:上皮细胞,8-15μg;成纤维细胞,5-7μg.需要但未提供的试剂:1. 氯仿2. 异丙醇3. 75%乙醇(depc处理水配制)4. 无rna酶水或0.5%sds溶液(制备无rna酶水:将水倒进无rna酶的玻璃容器中,加入depc至0.01%(v/v)放置过夜后高压灭菌). sds溶液必须用depc处理后高压灭菌的水配制.rna分离提取步骤:1．匀浆a. 组织每50-100mg组织加入1ml的trizol 试剂后用匀浆机或玻璃棒匀浆。

invitrogen_TRIZOL_中文说明书

TRIZOL® ReagentCat. No. 15596-026 Size: 100 mlStore at 2 to 8°C.警告：在与皮肤接触及吞咽有毒。

可导致烧伤。

与皮肤接触后，应立即用洗涤剂和大量水冲洗。

如感到身体不适，应就医（如需要，应出示本产品标签）。

本产品含有苯酚（108-95-2）和其他成分（NJTSRN 80100437-5000P）。

已经证明TRIZOL 在室温下可稳定保存12个月。

不过，我们建议在储存于2-8°C，以保证最佳性能。

描述：TRIzol试剂（美国专利号，5346994）是即用型细胞和组织总RNA提取试剂。

该试剂是一步法苯酚和异硫氰酸胍解决方案，是对Chomczynski和Sacchi开发的单步RNA提取法（1）的改善。

在匀质化或溶解样品中，TRIzol试剂可保持RNA的完整性，同时能破坏细胞及溶解细胞成分。

加入氯仿离心后，裂解液分离成水相和有机相。

RNA存在于水相。

水相转移后，RNA通过异丙醇沉淀回收。

移去水相后，样品中DNA和蛋白质可通过相继沉淀回收（2）。

用乙醇沉淀可从中间相得到DNA，加入异丙醇沉淀可从有机相得到蛋白质（2）。

与DNA的共纯化可能对不同样品得到的RNA的归一化有用。

此技术可完美应用于少量人类、动物、植物或细菌来源的组织（50-100毫克）和细胞（5×106），以及大量的组织（≥1 g）和细胞（>107）。

该TRIzol试剂方法简单，允许大量样本同时处理。

整个过程可在一小时内完成。

用TRIZOL提取总RNA可避免蛋白质和DNA 污染。

可用于Northern blot分析、斑点杂交、poly（A）+选择、体外翻译、RNA酶保护分析和分子克隆。

聚合酶链反应（PCR反应）中，当两条引物位于单个外显子时，推荐使用扩增级DNA酶I(Cat. No. 18068)处理分离出的RNA。

TRIzol试剂方便提取不同种类、不同分子大小的RNA。

Trizol使用说明书(TRIzol

TRIzol® ReagentCatalog Numbers Quantity Store at 2°C to 25°C 15596-026 100 mL15596-018 200 mLDescriptionTRIzol® Reagent is a ready-to-use reagent, designed to isolate high quality total RNA (as well as DNA and proteins) from cell and tissue samples of human, animal, plant, yeast, or bacterial origin, within one hour. TRIzol® Reagent is a monophasic solution ofphenol, guanidine isothiocyanate, and other proprietary components which facilitate the isolation of a variety of RNA species of large or small molecular size. TRIzol® Reagent maintains the integrity of the RNA due to highly effective inhibition of RNaseactivity while disrupting cells and dissolving cell components during sample homogenization. TRIzol® Reagent allows forsimultaneous processing of a large number of samples, and is an improvement to the single-step RNA isolation method developed by Chomcynski and Sacchi (Chomczynski & Sacchi, 1987).TRIzol® Reagent allows the user to perform sequential precipitation of RNA, DNA, and proteins from a single sample(Chomczynski, 1993). After homogenizing the sample with TRIzol® Reagent, chloroform is added, and the homogenate is allowed to separate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). RNA is precipitated from the aqueous layer with isopropanol. DNA is precipitated from the interphase/organic layer with ethanol. Protein is precipitated from the phenol-ethanol supernatant by isopropanol precipitation. The precipitated RNA, DNA, or protein is washed to remove impurities, and then resuspended for use in downstream applications.•Isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assay, and molecular cloning.•Isolated DNA can be used in PCR, Restriction Enzyme digestion, and Southern Blots.•Isolated protein can be used for Western Blots, recovery of some enzymatic activity, and some immunoprecipitation. CautionTRIzol® Reagent contains phenol (toxic and corrosive) and guanidine isothiocyanate (an irritant), and may be a health hazard if not handled properly. Always work with TRIzol® Reagent in a fume hood, and always wear a lab coat, gloves and safety glasses. Contact your Environmental Heath and Safety (EH&S) department for proper work and disposal guidelines. Avoid direct contact with TRIzol® Reagent, because contact to skin, eyes, or respiratory tract may cause chemical burns to the exposed area. If contact to skin or eyes occurs, immediately wash the exposed area with copious amounts of water for 15 minutes and seek medical attention if necessary. If you inhale vapors, move to fresh air and seek medical attention if necessary. For more information, refer to the TRIzol® Reagent SDS (Safety Data Sheet), available from our web site at /support. Contents and StorageTRIzol® Reagent is supplied in 100 mL (Cat. no. 15596-026) or 200 mL (Cat. no. 15596-018) volumes, and shipped at room temperature. Upon receipt, store TRIzol® Reagent at room temperature. TRIzol® Reagent is stable for 12 months when properly stored.Intended UseFor research use only. Not intended for human or animal diagnostic or therapeutic uses.Materials NeededThe following additional materials are needed, but not supplied for the isolation of RNA, DNA or proteins.Part no. 15596026.PPS MAN0001271Rev. Date: 13 Dec 2012For support, visit /support or email techsupport@. To reorder, visit Preparing Samples Homogenizing samples 1.at room temperature according to the table below. Thesample volume should not exceed 10% of the volume ofTRIzol® Reagent used for homogenization. Be sure to usethe indicated amount of TRIzol® Reagent, because aninsufficient volume can result in DNA contamination ofisolated RNA.2.(Optional) When preparing samples with high content offat, proteins, polysaccharides, or extracellular material(e.g., muscle, fat tissue, or tuberous plant material), anadditional isolation step may be required to removeinsoluble material from the samples.Note: Do not perform this additional isolation step if youare performing subsequent DNA isolation on your sample.3.Proceed to Phase separation, or store the homogenizedsample. Homogenized samples can be stored at roomtemperature for several hours, or at –60 to –70°C for at leastone month.Incubate the homogenized sample (see Homogenizingsamples) for 5 minutes at room temperature to permitcomplete dissociation of the nucleoprotein complex.2.Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagentused for homogenization. Cap the tube securely.3.Shake tube vigorously by hand for 15 seconds.4.Incubate for 2–3 minutes at room temperature.5.Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.Note: The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upperaqueous phase. RNA remains exclusively in the aqueous phase.The upper aqueous phase is ~50% of the total volume.6.Remove the aqueous phase of the sample by angling thetube at 45° and pipetting the solution out. Avoid drawingany of the interphase or organic layer into the pipette whenremoving the aqueous phase.7.Place the aqueous phase into a new tube and proceed tothe RNA Isolation Procedure.8.Save the interphase and organic phenol-chloroform phaseif isolation of DNA or protein is desired. See DNAIsolation Procedure and Protein Isolation Procedure fordetails. The organic phase can be stored at 4°C overnight.RNA Isolation ProcedureAlways use the appropriate precautions to avoid RNasecontamination when preparing and handling RNA.RNA precipitation1.(Optional) When precipitating RNA from small samplequantities (<106 cells or <10 mg tissue), a dd 5–10 µg ofRNase-free glycogen as a carrier to the aqueous phase.Note: Glycogen is co-precipitated with the RNA, but doesnot inhibit first-strand synthesis at concentrations≤4 mg/mL, and does not inhibit PCR.2.Add 0.5 mL of 100% isopropanol to the aqueous phase, per1 mL of TRIzol® Reagent used for homogenization.3.Incubate at room temperature for 10 minutes.4.Centrifuge at 12,000 × g for 10 minutes at 4°C.Note: The RNA is often invisible prior to centrifugation,and forms a gel-like pellet on the side and bottom of thetube.5.Proceed to RNA wash.RNA wash1.Remove the supernatant from the tube, leaving only theRNA pellet.2.Wash the pellet, with 1 mL of 75% ethanol per 1 mL ofTRIzol® Reagent used in the initial homogenization.Note: The RNA can be stored in 75% ethanol at least 1 yearat –20°C, or at least 1 week at 4°C.3.Vortex the sample briefly, then centrifuge the tube at7500 × g for 5 minutes at 4°C. Discard the wash.4.Vacuum or air dry the RNA pellet for 5–10 minutes. Do notdry the pellet by vacuum centrifuge.Note: Do not allow the RNA to dry completely, becausethe pellet can lose solubility. Partially dissolved RNAsamples have an A260/280 ratio <1.6.5.Proceed to RNA resuspension.RNA resuspension1.Resuspend the RNA pellet in RNase-free water or0.5% SDS solution (20–50 μL) by passing the solution upand down several times through a pipette tip.Note: Do not dissolve the RNA in 0.5% SDS if it is to beused in subsequent enzymatic reactions.2.Incubate in a water bath or heat block set at 55–60°C for10–15 minutes.3.Proceed to downstream application, or store at –70°C. DNA Isolation ProcedureDNA is isolated from the interphase and phenol-chloroform layer saved from the Phase separation step.DNA precipitation1.Remove any remaining aqueous phase overlying theinterphase. This is critical for the quality of the isolatedDNA.2.Add 0.3 mL of 100% ethanol per of 1 mL TRIzol® Reagentused for the initial homogenization.3.Cap the tube and invert the sample several times to mix.4.Incubate samples for 2–3 minutes at room temperature.5.Centrifuge the tube at 2000 × g for 5 minutes at 4°C topellet the DNA.6.Remove the phenol-ethanol supernatant and save it in anew tube if protein isolation is desired. The supernatantcan be stored at –70°C for several months.7.Proceed with the DNA wash step using the DNA pellet. DNA wash1.Wash the DNA pellet with 1 mL of sodium citrate/ ethanolsolution (0.1 M sodium citrate in 10% ethanol, pH 8.5) per1 mL of TRIzol® Reagent used for the initialhomogenization.2.Incubate for 30 minutes at room temperature. Mixoccasionally by gentle inversion.Note: The DNA can be stored in sodium citrate/ethanolsolution at least 2 hours.3.Centrifuge at 2000 × g for 5 minutes at 4°C. Remove anddiscard supernatant.4.Repeat wash (steps 1–3), once.Note: Repeat wash twice for large DNA pellets (>200 µg).5.Add 1.5–2 mL 75% ethanol per 1 mL of TRIzol® Reagentused for the initial homogenization.Note: DNA samples may be stored in 75% ethanol at 4°Cfor several months.6.Incubate for 10–20 minutes at room temperature. Mix thetube occasionally by gentle inversion.7.Centrifuge at 2000 × g for 5 minutes at 4°C. Remove anddiscard supernatant.8.Air or vacuum dry the DNA pellet for 5–10 minutes. Donot allow the pellet to dry out. Do not dry the pellet byvacuum centrifuge.9.Proceed to the DNA resuspension step.DNA resuspensionResuspend the DNA in 8mM NaOH at a concentration of0.2–0.3 µg/µL.1.Add 0.3–0.6 mL of 8mM NaOH per 50–70 mg of tissue,or per 1 × 107 cells.Note: Resuspending the DNA is a mild base is highlyrecommended because isolated DNA does notresuspend well in water or Tris buffer.2.Remove any insoluble material by centrifuging thesample at 12,000 × g for 10 minutes at 4°C.3.Transfer the supernatant containing the DNA to a newtube. Adjust pH as needed with HEPES and proceed to downstream application of choice. The DNA can bestored overnight at 4°C, but for long-term storage adjust to pH 7–8 with HEPES, and add 1 mM EDTA. Store at4°C or –20°C.Determining Yield of RNA and DNAUse absorbance of RNA and DNA at 260 nm and 280 nm to determine concentration.Expected yieldsThe table below presents typical yields of RNA (A260/280 of>1.8) and DNA (A260/280 of 1.6–1.8) from various starting materials.Protein Isolation ProcedureProteins are isolated from the phenol-ethanol supernatant layer left over after the DNA precipitation step. Isolate the protein using either Protein precipitation OR Protein dialysis. Protein precipitation1.Add 1.5 mL of isopropanol to the phenol-ethanolsupernatant per of 1 mL TRIzol® Reagent used for theinitial homogenization.2.Incubate samples for 10 minutes at room temperature.3.Centrifuge at 12,000 × g for 10 minutes at 4°C to pellet theprotein. Remove and discard the supernatant.4.Proceed to the Protein wash step with the remainingprotein pellet.Protein wash1.Prepare a wash solution consisting of 0.3 M guanidinehydrochloride in 95% ethanol.2.Wash the protein pellet with 2 mL of the wash solution per1 mL of TRIzol® Reagent used for the initial homogenization.3.Incubate for 20 minutes at room temperature.Note: Protein samples may be stored in 0.3 M guanidinehydrochloride-95% ethanol for at least one month at 4°C orfor at least one year at –20°C.4.Centrifuge at 7500 × g for 5 minutes at 4°C. Remove anddiscard the wash solution.5.Repeat steps 2–4, two more times.6.Add 2 mL of 100% ethanol to protein pellet after the thirdwash and vortex.7.Incubate for 20 minutes at room temperature.8.Centrifuge at 7500 × g for 5 minutes at 4°C. Remove anddiscard ethanol wash.9.Air dry the protein pellet for 5–10 minutes. Do not allow thepellet to dry out.10.Proceed to the Protein resuspension step.Protein resuspension1.Add 1% SDS to the protein pellet (200 μL) and pipet up anddown until the protein is resuspend.Note: To completely dissolve the protein pellet, you may need to incubate the sample at 50°C in a water bath or heat block.2.Centrifuge at 10,000 × g for 10 minutes at 4°C to sediment anyinsoluble material.3.Transfer the supernatant containing the protein to a new tubeand proceed to downstream application of choice, or store the sample at –20°C. Protein resuspension, continuedPoor solubility of the pellet in SDS can occur, because the solubilityof specific classes of proteins differs with different solvents. If the protein pellet is insoluble in SDS, the following alternative solvents(Hummon et. al., 2007) may be required to solubilize the pellet: •1% SDS and 62.5 mM sarkosyl at pH 8.0–8.8•9.5 M urea and 2% CHAPS, pH 9.1•250mM glycerol, 10mM TEA, and 4% CHAPS•2% diethylamine•10M UreaProtein dialysis1.Load the phenol-ethanol supernatant into the dialysismembrane.Note: The phenol-ethanol solution can dissolve some types ofdialysis membranes (e.g., cellulose ester). Test dialysis tubingwith the membrane to assess compatibility before starting.2.Dialyze the sample against 3 changes of 0.1% SDS at 4°C. Makethe first change of solution after 16 hours, the second change4 hours later (at 20 hours), and the final change 2 hours later (at22 hours).Note: 0.1% SDS is required to resolubilize the proteins from the pellet; a lower concentration of SDS is insufficient. If desired,the SDS can be diluted after solubilization.3.Centrifuge the dialysate at 10,000 × g for 10 minutes at 4°C.Proteins are located in the clear supernatant.4.Transfer supernatant to a new tube and proceed to downstreamapplication, or store the sample at –20°C.5.(Optional) Solubilize the pellet by adding 100 μL of 1% SDS and100 μL of 8 M urea.Determining Yield of ProteinMeasure protein concentration by Bradford assay (SDSconcentration must be <0.1%).TroubleshootingReferences:Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532-537Chomczynski, P., and Sacchi, N. (1987) Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 162, 156-159Hummon, A. B., Lim S. R., Difilippantonio, M. J., Ried, T. (2007) Isolation and solubilization of proteins after TRIzol® extraction of RNA and DNA from patient material following prolonged storage.BioTechniques 42, 467-472Limited Use Label License No. 358: Research Use Only: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial services of any kind, including, without limitation, reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@ or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.©2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.TRIzol® is a registered trademark of Molecular Research Center, Inc.。

trizol提取RNA说明书

Trizol 使用说明书一、分离纯化的基本原理研究基因的表达和调控时常常要从组织和细胞中分离和纯化 RNA。

RNA 质量的高低常常影响cDNA 库，RT-PCR 和 Northern Blot 等分子生物学实验的成败。

Trizol 是一种新型总 RNA 抽提试剂，内含异硫氰酸胍等物质，能迅速破碎细胞，抑制细胞释放出的核酸酶。

二、用户需自备的试剂和材料无水乙醇、氯仿、Glycogen（可能需要）、 1.5ml Eppendorf 管（RNase-free）、 Tips（RNase-free）三、准备工作RNase 酶非常稳定，是导致 RNA 降解最主要的物质。

它在一些极端的条件可以暂时失活，但限制因素去除后有迅速复性。

用常规的高温高压蒸气灭菌方法和蛋白抑制剂都不能是 RNase 完全失活。

它广泛存在于人的皮肤上，因此，在与 RNA 制备有关的分子生物学实验时，必须戴手套。

RNase 的又一污染源是取液器，根据取液器制造商的要求对取液器进行处理。

一般情况下采用用DEPC 配制的 70%乙醇擦洗取液器的内部和外部，基本达到要求。

取 RNase-free 的物品时必须戴手套。

1、料制品的处理尽可能使用无菌，一次性塑料制品，已标明 RNase-Free 的塑料制品，如没有开封使用过通常没有必要再次处理。

对于国产塑料制品，原则上都必须处理方可使用。

处理步骤如下：1）在玻璃烧杯中注入去离子水，加入 DEPC 使 DEPC 的终浓度为 0.1%。

注意：DEPC 为剧毒物，活性很强，小心在通风柜中使用。

2）处理的塑料制品放入一个可以高温灭菌的容器中，注入 DEPC 水溶液，使塑料制品的所有部分都浸泡到溶液中。

3）在通风柜中室温处理过夜。

4）将 DEPC 水溶液小心倒到废液瓶中，用铝箔封住含已 DEPC 水处理过的塑料制品的烧杯，高温高压蒸气灭菌至少 30 分钟。

5）烘箱用合适的温度烘拷至干燥。

置于干净处备用。

2、璃玻和金属物品250°C 烘烤 3 小时以上。

RNA提取trizol试剂盒说明书

注：各种样品的最大使用量（1ml Trizol）操作步骤：1. 在液氮中将组织(单头飞虱)研磨成粉末，趁液氮尚未挥发光时，将粉末转移到1.5ml离心管中。

细胞经计数后直接加入离心管，然后5000rpm室温离心去上清。

每100mg组织或5×106个细胞加1ml的Trizol。

注意：如果组织量（1-10mg）或细胞数很少（1×102-1×104），在样品中加入800μl的Trizol，用枪头反复抽吸混匀，再加入糖原（终浓度为250μg/ml），剧烈振荡或用匀浆器匀浆。

2. 用1ml针筒，26-G号（6#）针头抽吸匀浆液两次以剪切基因组DNA，然后直接从针筒中将样品转移到无菌1.5-ml离心管中。

3. 加入200μl氯仿/异戊醇（24:1）或氯仿，剧烈振荡混匀30秒。

4. 台式离心机上，12000rpm，室温离心5分钟。

5. 将上清液小心转移到RNase-free 1.5ml 离心管中，加入等体积的异丙醇，室温下放置5分钟。

（注意：不要吸取任何中间层物质，否则会出现染色体DNA污染）6. 台式离心机上，12000rpm，室温离心5分钟。

7．小心移去上清液，防止RNA沉淀丢失。

8. 用70%酒精洗涤两次，每次700μl，12000rpm，室温离心2分钟。

9. 尽可能彻底地吸走上清，防止RNA沉淀丢失。

10. 真空离心干燥3-5分钟，或放在室温下使酒精完全挥发。

11. 沉淀用30-50μl DEPC-H2O溶解。

如发现沉淀难溶，68℃处理10分钟。

对于胰腺，肾等组织中RNase含量很高，沉淀用100%去离子甲酰胺溶解。

12．DNA的分析和定量：（1）测定样品在260nm和280nm的吸收值确定RNA的质量。

按1 OD=40μg RNA计算RNAD的产率：OD260/280在1.8-2.0视为抽提的RNA纯度很高。

若需精确量化，只有浓度在4μg/ml以上的样品适于用光度计测定。

Trizol中文说明书

Trizol 中文说明书TRIzol 试剂是一种从组织及细胞中提取总RNA的专用试剂。

这种试剂是一种酚和硫氰酸胍的单相溶液，是将Chomczynski 和Sacchi发明的一步RNA提取法的改进。

在样品匀浆或分析过程中，TRIzol 试剂可在分裂细胞及溶解胞膜的同时保持RNA的完整。

在匀浆后加入氯仿，可将溶液分为水相和有机相。

RNA均在水相中。

吸取水相加入异丙醇，得到RNA沉淀。

去除水相后，样品中的DNA及蛋白质可通过进一步连续的沉淀而得到。

中间相加入乙醇可沉淀DNA而有机相加入异丙醇则能沉淀出蛋白质。

DNA的再纯化可能对标化样品的RNA产量有作用。

2．这一技术可以用于人，动物，植物或细菌株的微量组织（50—100mg）及细胞（5×106），也可以用于大量检材（≥1g）及细胞(﹥107).该方法简便易行,可用于同时检测大量样品,全部过程1小时内可以完成.用TRIzol 试剂分离的RNA 没有DNA及蛋白质成分.这种RNA可用于Northern印迹分析,斑点杂交,多聚(A)+检出及Vitro转移, RNA酶蛋白分析及分子克隆.用于PCR反应时,当两个引物位于同一外显子时应用扩增特异DNA酶I处理提取出的RNA.3． TRIzol 试剂可用于分子量从大到小的各种RNA的提取.例如,大鼠肝脏提取出的RNA,经琼脂糖凝胶电泳,EB染色后可显现出7kb和15kb的大分子量的RNA谱带(包括mRNA和hnRNA), 位于～5kb(28s)和～2kb (18s)的两条主要的核糖体RNA谱带及大小在0.1kb-0.3kb之间的低分子量RNA(tRNA,5s).分离出的RNA当溶于双蒸水中时A260/280为1.6-1.8之间.每毫克组织所能提取的RNA量约为:肝和脾,6-10μg;肾,3-4μg;骨骼肌和脑,1-5μg;胎盘,1-4μg.从1×106培养细胞中提取的RNA量约为:上皮细胞,8-15μg;成纤维细胞,5-7μg.需要但未提供的试剂:1. 氯仿2. 异丙醇3. 75%乙醇(DEPC处理水配制)4. 无RNA酶水或0.5%SDS溶液(制备无RNA酶水:将水倒进无RNA酶的玻璃容器中,加入DEPC 至0.01%(v/v)放置过夜后高压灭菌). SDS溶液必须用DEPC处理后高压灭菌的水配制.RNA分离提取步骤:1．匀浆a. 组织每50-100mg组织加入1ml的TRIzol 试剂后用匀浆机或玻璃棒匀浆。

RNA提取Trizol 使用说明书

Trizol使用说明书一、分离纯化的基本原理研究基因的表达和调控时常常要从组织和细胞中分离和纯化RNA。

RNA质量的高低常常影响cDNA库，RT-PCR和Northern Blot等分子生物学实验的成败。

Trizol是一种新型总RNA抽提试剂，内含异硫氰酸胍等物质，能迅速破碎细胞，抑制细胞释放出的核酸酶。

二、用户需自备的试剂和材料无水乙醇、氯仿、Glycogen（可能需要）、 1.5ml Eppendorf管（RNase-free）、 Tips （RNase-free）三、准备工作RNase酶非常稳定，是导致RNA降解最主要的物质。

它在一些极端的条件可以暂时失活，但限制因素去除后有迅速复性。

用常规的高温高压蒸气灭菌方法和蛋白抑制剂都不能是RNase完全失活。

它广泛存在于人的皮肤上，因此，在与RNA制备有关的分子生物学实验时，必须戴手套。

RNase的又一污染源是取液器，根据取液器制造商的要求对取液器进行处理。

一般情况下采用用DEPC配制的70%乙醇擦洗取液器的内部和外部，基本达到要求。

取RNase-free的物品时必须戴手套。

1、料制品的处理尽可能使用无菌，一次性塑料制品，已标明RNase-Free 的塑料制品，如没有开封使用过通常没有必要再次处理。

对于国产塑料制品，原则上都必须处理方可使用。

处理步骤如下：1）在玻璃烧杯中注入去离子水，加入DEPC使DEPC的终浓度为0.1%。

注意：DEPC为剧毒物，活性很强，小心在通风柜中使用。

2）处理的塑料制品放入一个可以高温灭菌的容器中，注入DEPC水溶液，使塑料制品的所有部分都浸泡到溶液中。

3）在通风柜中室温处理过夜。

4）将DEPC水溶液小心倒到废液瓶中，用铝箔封住含已DEPC水处理过的塑料制品的烧杯，高温高压蒸气灭菌至少30分钟。

5）烘箱用合适的温度烘拷至干燥。

置于干净处备用。

2、璃玻和金属物品250°C烘烤3小时以上。

四、从组织中提取总RNA1)液氮研磨，组织块直接放入研钵中，加入少量液氮，迅速研磨，待组织变软，再加少量液氮，再研磨，如此三次，按50-100mg组织/ml Trizol加入Trizol，转入离心管进行第2步操作。

诺维赞trizol说明书

诺维赞trizol说明书一、药物名称商品名：诺维赞（Novalzone）通用名：trizol二、药物成分每片（包或瓶）含有主要成分trizol 500mg。

三、适应症诺维赞（trizol）适用于治疗以下感染性疾病：1.呼吸道感染2.泌尿系统感染3.皮肤和软组织感染4.肺炎5.腹部感染6.中耳炎7.骨关节感染8.表皮感染9.尿道感染10.盆腔炎四、禁忌症1.对磺胺类药物过敏者2.肾功能不全或肾衰竭患者3.儿童（特别是6岁以下）五、用法和用量成人一般剂量为每日2-4次，每次服用1片（500mg）。

儿童用量需根据年龄和体重进行确定，以避免过量。

应按照医生的指示准确服用。

六、注意事项1.使用前请阅读本说明书，并请遵循医生的建议。

4.患有肝功能异常的患者应在医生指导下使用。

5.患有糖尿病的患者请监测血糖，因为该药物可能会影响血糖水平。

6.使用过程中如出现恶心、呕吐、头晕等不适症状，应及时就医。

七、不良反应1.常见的不良反应包括胃肠道不适，如恶心、呕吐、腹泻、腹痛等。

2.偶尔可能出现过敏反应，如皮疹、红肿、呼吸困难等。

八、药物相互作用1.请告诉医生您正在使用的所有药物，包括非处方药、补品和保健品。

2. trizol可能与一些药物相互作用，增加药物的毒性或降低疗效。

请遵循医生的建议。

九、贮藏方式1.请将药物放在干燥、避光的地方，远离儿童。

2.药物过期后请勿使用。

十、生产厂家本药品由诺维赞制药有限公司生产。

Trizol使用说明书(TRIzol

TRIzol® ReagentCatalog Numbers Quantity Store at 2°C to 25°C 15596-026 100 mL15596-018 200 mLDescriptionTRIzol® Reagent is a ready-to-use reagent, designed to isolate high quality total RNA (as well as DNA and proteins) from cell and tissue samples of human, animal, plant, yeast, or bacterial origin, within one hour. TRIzol® Reagent is a monophasic solution ofphenol, guanidine isothiocyanate, and other proprietary components which facilitate the isolation of a variety of RNA species of large or small molecular size. TRIzol® Reagent maintains the integrity of the RNA due to highly effective inhibition of RNaseactivity while disrupting cells and dissolving cell components during sample homogenization. TRIzol® Reagent allows forsimultaneous processing of a large number of samples, and is an improvement to the single-step RNA isolation method developed by Chomcynski and Sacchi (Chomczynski & Sacchi, 1987).TRIzol® Reagent allows the user to perform sequential precipitation of RNA, DNA, and proteins from a single sample(Chomczynski, 1993). After homogenizing the sample with TRIzol® Reagent, chloroform is added, and the homogenate is allowed to separate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). RNA is precipitated from the aqueous layer with isopropanol. DNA is precipitated from the interphase/organic layer with ethanol. Protein is precipitated from the phenol-ethanol supernatant by isopropanol precipitation. The precipitated RNA, DNA, or protein is washed to remove impurities, and then resuspended for use in downstream applications.•Isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assay, and molecular cloning.•Isolated DNA can be used in PCR, Restriction Enzyme digestion, and Southern Blots.•Isolated protein can be used for Western Blots, recovery of some enzymatic activity, and some immunoprecipitation. CautionTRIzol® Reagent contains phenol (toxic and corrosive) and guanidine isothiocyanate (an irritant), and may be a health hazard if not handled properly. Always work with TRIzol® Reagent in a fume hood, and always wear a lab coat, gloves and safety glasses. Contact your Environmental Heath and Safety (EH&S) department for proper work and disposal guidelines. Avoid direct contact with TRIzol® Reagent, because contact to skin, eyes, or respiratory tract may cause chemical burns to the exposed area. If contact to skin or eyes occurs, immediately wash the exposed area with copious amounts of water for 15 minutes and seek medical attention if necessary. If you inhale vapors, move to fresh air and seek medical attention if necessary. For more information, refer to the TRIzol® Reagent SDS (Safety Data Sheet), available from our web site at /support. Contents and StorageTRIzol® Reagent is supplied in 100 mL (Cat. no. 15596-026) or 200 mL (Cat. no. 15596-018) volumes, and shipped at room temperature. Upon receipt, store TRIzol® Reagent at room temperature. TRIzol® Reagent is stable for 12 months when properly stored.Intended UseFor research use only. Not intended for human or animal diagnostic or therapeutic uses.Materials NeededThe following additional materials are needed, but not supplied for the isolation of RNA, DNA or proteins.Part no. 15596026.PPS MAN0001271Rev. Date: 13 Dec 2012For support, visit /support or email techsupport@. To reorder, visit Preparing Samples Homogenizing samples 1.at room temperature according to the table below. Thesample volume should not exceed 10% of the volume ofTRIzol® Reagent used for homogenization. Be sure to usethe indicated amount of TRIzol® Reagent, because aninsufficient volume can result in DNA contamination ofisolated RNA.2.(Optional) When preparing samples with high content offat, proteins, polysaccharides, or extracellular material(e.g., muscle, fat tissue, or tuberous plant material), anadditional isolation step may be required to removeinsoluble material from the samples.Note: Do not perform this additional isolation step if youare performing subsequent DNA isolation on your sample.3.Proceed to Phase separation, or store the homogenizedsample. Homogenized samples can be stored at roomtemperature for several hours, or at –60 to –70°C for at leastone month.Incubate the homogenized sample (see Homogenizingsamples) for 5 minutes at room temperature to permitcomplete dissociation of the nucleoprotein complex.2.Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagentused for homogenization. Cap the tube securely.3.Shake tube vigorously by hand for 15 seconds.4.Incubate for 2–3 minutes at room temperature.5.Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.Note: The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upperaqueous phase. RNA remains exclusively in the aqueous phase.The upper aqueous phase is ~50% of the total volume.6.Remove the aqueous phase of the sample by angling thetube at 45° and pipetting the solution out. Avoid drawingany of the interphase or organic layer into the pipette whenremoving the aqueous phase.7.Place the aqueous phase into a new tube and proceed tothe RNA Isolation Procedure.8.Save the interphase and organic phenol-chloroform phaseif isolation of DNA or protein is desired. See DNAIsolation Procedure and Protein Isolation Procedure fordetails. The organic phase can be stored at 4°C overnight.RNA Isolation ProcedureAlways use the appropriate precautions to avoid RNasecontamination when preparing and handling RNA.RNA precipitation1.(Optional) When precipitating RNA from small samplequantities (<106 cells or <10 mg tissue), a dd 5–10 µg ofRNase-free glycogen as a carrier to the aqueous phase.Note: Glycogen is co-precipitated with the RNA, but doesnot inhibit first-strand synthesis at concentrations≤4 mg/mL, and does not inhibit PCR.2.Add 0.5 mL of 100% isopropanol to the aqueous phase, per1 mL of TRIzol® Reagent used for homogenization.3.Incubate at room temperature for 10 minutes.4.Centrifuge at 12,000 × g for 10 minutes at 4°C.Note: The RNA is often invisible prior to centrifugation,and forms a gel-like pellet on the side and bottom of thetube.5.Proceed to RNA wash.RNA wash1.Remove the supernatant from the tube, leaving only theRNA pellet.2.Wash the pellet, with 1 mL of 75% ethanol per 1 mL ofTRIzol® Reagent used in the initial homogenization.Note: The RNA can be stored in 75% ethanol at least 1 yearat –20°C, or at least 1 week at 4°C.3.Vortex the sample briefly, then centrifuge the tube at7500 × g for 5 minutes at 4°C. Discard the wash.4.Vacuum or air dry the RNA pellet for 5–10 minutes. Do notdry the pellet by vacuum centrifuge.Note: Do not allow the RNA to dry completely, becausethe pellet can lose solubility. Partially dissolved RNAsamples have an A260/280 ratio <1.6.5.Proceed to RNA resuspension.RNA resuspension1.Resuspend the RNA pellet in RNase-free water or0.5% SDS solution (20–50 μL) by passing the solution upand down several times through a pipette tip.Note: Do not dissolve the RNA in 0.5% SDS if it is to beused in subsequent enzymatic reactions.2.Incubate in a water bath or heat block set at 55–60°C for10–15 minutes.3.Proceed to downstream application, or store at –70°C. DNA Isolation ProcedureDNA is isolated from the interphase and phenol-chloroform layer saved from the Phase separation step.DNA precipitation1.Remove any remaining aqueous phase overlying theinterphase. This is critical for the quality of the isolatedDNA.2.Add 0.3 mL of 100% ethanol per of 1 mL TRIzol® Reagentused for the initial homogenization.3.Cap the tube and invert the sample several times to mix.4.Incubate samples for 2–3 minutes at room temperature.5.Centrifuge the tube at 2000 × g for 5 minutes at 4°C topellet the DNA.6.Remove the phenol-ethanol supernatant and save it in anew tube if protein isolation is desired. The supernatantcan be stored at –70°C for several months.7.Proceed with the DNA wash step using the DNA pellet. DNA wash1.Wash the DNA pellet with 1 mL of sodium citrate/ ethanolsolution (0.1 M sodium citrate in 10% ethanol, pH 8.5) per1 mL of TRIzol® Reagent used for the initialhomogenization.2.Incubate for 30 minutes at room temperature. Mixoccasionally by gentle inversion.Note: The DNA can be stored in sodium citrate/ethanolsolution at least 2 hours.3.Centrifuge at 2000 × g for 5 minutes at 4°C. Remove anddiscard supernatant.4.Repeat wash (steps 1–3), once.Note: Repeat wash twice for large DNA pellets (>200 µg).5.Add 1.5–2 mL 75% ethanol per 1 mL of TRIzol® Reagentused for the initial homogenization.Note: DNA samples may be stored in 75% ethanol at 4°Cfor several months.6.Incubate for 10–20 minutes at room temperature. Mix thetube occasionally by gentle inversion.7.Centrifuge at 2000 × g for 5 minutes at 4°C. Remove anddiscard supernatant.8.Air or vacuum dry the DNA pellet for 5–10 minutes. Donot allow the pellet to dry out. Do not dry the pellet byvacuum centrifuge.9.Proceed to the DNA resuspension step.DNA resuspensionResuspend the DNA in 8mM NaOH at a concentration of0.2–0.3 µg/µL.1.Add 0.3–0.6 mL of 8mM NaOH per 50–70 mg of tissue,or per 1 × 107 cells.Note: Resuspending the DNA is a mild base is highlyrecommended because isolated DNA does notresuspend well in water or Tris buffer.2.Remove any insoluble material by centrifuging thesample at 12,000 × g for 10 minutes at 4°C.3.Transfer the supernatant containing the DNA to a newtube. Adjust pH as needed with HEPES and proceed to downstream application of choice. The DNA can bestored overnight at 4°C, but for long-term storage adjust to pH 7–8 with HEPES, and add 1 mM EDTA. Store at4°C or –20°C.Determining Yield of RNA and DNAUse absorbance of RNA and DNA at 260 nm and 280 nm to determine concentration.Expected yieldsThe table below presents typical yields of RNA (A260/280 of>1.8) and DNA (A260/280 of 1.6–1.8) from various starting materials.Protein Isolation ProcedureProteins are isolated from the phenol-ethanol supernatant layer left over after the DNA precipitation step. Isolate the protein using either Protein precipitation OR Protein dialysis. Protein precipitation1.Add 1.5 mL of isopropanol to the phenol-ethanolsupernatant per of 1 mL TRIzol® Reagent used for theinitial homogenization.2.Incubate samples for 10 minutes at room temperature.3.Centrifuge at 12,000 × g for 10 minutes at 4°C to pellet theprotein. Remove and discard the supernatant.4.Proceed to the Protein wash step with the remainingprotein pellet.Protein wash1.Prepare a wash solution consisting of 0.3 M guanidinehydrochloride in 95% ethanol.2.Wash the protein pellet with 2 mL of the wash solution per1 mL of TRIzol® Reagent used for the initial homogenization.3.Incubate for 20 minutes at room temperature.Note: Protein samples may be stored in 0.3 M guanidinehydrochloride-95% ethanol for at least one month at 4°C orfor at least one year at –20°C.4.Centrifuge at 7500 × g for 5 minutes at 4°C. Remove anddiscard the wash solution.5.Repeat steps 2–4, two more times.6.Add 2 mL of 100% ethanol to protein pellet after the thirdwash and vortex.7.Incubate for 20 minutes at room temperature.8.Centrifuge at 7500 × g for 5 minutes at 4°C. Remove anddiscard ethanol wash.9.Air dry the protein pellet for 5–10 minutes. Do not allow thepellet to dry out.10.Proceed to the Protein resuspension step.Protein resuspension1.Add 1% SDS to the protein pellet (200 μL) and pipet up anddown until the protein is resuspend.Note: To completely dissolve the protein pellet, you may need to incubate the sample at 50°C in a water bath or heat block.2.Centrifuge at 10,000 × g for 10 minutes at 4°C to sediment anyinsoluble material.3.Transfer the supernatant containing the protein to a new tubeand proceed to downstream application of choice, or store the sample at –20°C. Protein resuspension, continuedPoor solubility of the pellet in SDS can occur, because the solubilityof specific classes of proteins differs with different solvents. If the protein pellet is insoluble in SDS, the following alternative solvents(Hummon et. al., 2007) may be required to solubilize the pellet: •1% SDS and 62.5 mM sarkosyl at pH 8.0–8.8•9.5 M urea and 2% CHAPS, pH 9.1•250mM glycerol, 10mM TEA, and 4% CHAPS•2% diethylamine•10M UreaProtein dialysis1.Load the phenol-ethanol supernatant into the dialysismembrane.Note: The phenol-ethanol solution can dissolve some types ofdialysis membranes (e.g., cellulose ester). Test dialysis tubingwith the membrane to assess compatibility before starting.2.Dialyze the sample against 3 changes of 0.1% SDS at 4°C. Makethe first change of solution after 16 hours, the second change4 hours later (at 20 hours), and the final change 2 hours later (at22 hours).Note: 0.1% SDS is required to resolubilize the proteins from the pellet; a lower concentration of SDS is insufficient. If desired,the SDS can be diluted after solubilization.3.Centrifuge the dialysate at 10,000 × g for 10 minutes at 4°C.Proteins are located in the clear supernatant.4.Transfer supernatant to a new tube and proceed to downstreamapplication, or store the sample at –20°C.5.(Optional) Solubilize the pellet by adding 100 μL of 1% SDS and100 μL of 8 M urea.Determining Yield of ProteinMeasure protein concentration by Bradford assay (SDSconcentration must be <0.1%).TroubleshootingReferences:Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532-537Chomczynski, P., and Sacchi, N. (1987) Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 162, 156-159Hummon, A. B., Lim S. R., Difilippantonio, M. J., Ried, T. (2007) Isolation and solubilization of proteins after TRIzol® extraction of RNA and DNA from patient material following prolonged storage.BioTechniques 42, 467-472Limited Use Label License No. 358: Research Use Only: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial services of any kind, including, without limitation, reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@ or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.©2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.TRIzol® is a registered trademark of Molecular Research Center, Inc.。

trizol LS 试剂使用中文说明书

TRIzol LS 试剂(ambion)（中文说明书）货号规格室温贮存10296-010 100ml10296-028 200ml产品描述TRIzol LS试剂适用于液体样品，如人、动植物、酵母、细菌或者病毒来源的液体样品，提取高质量的总RNA（DNA和蛋白质也可以）全过程只需1小时。

TRIzol LS试剂里含有酚、异硫氰酸胍和其它成分，由于在摇匀样品过程中能很有效地抑制RNase活性，所以TRIzol LS试剂能维持RNA完整性。

TRIzol LS试剂允许同时处理大量的样品。

TRIzol LS试剂能从一个样品里有序分离提取RNA、DNA和蛋白质。

TRIzol LS试剂均质化样品后，加入氯仿，能看见分层现象，上层是透明的含RNA的水相，中间层，下层是红色的有机相（含有DNA和蛋白质）。

水相RNA用异丙醇能沉淀分离；中间层或者下层中的DNA用乙醇沉淀分离；异丙醇沉淀能使蛋白质从酚-醇相的上清液中沉淀出来。

沉淀出的RNA、DNA 和蛋白质洗脱杂质，重悬后用于下游。

分离的RNA可以用于RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assay, and molecular cloning 分离的DNA可以用于PCR, and Restriction Enzyme digestion, and Southern Blots.分离的蛋白质可以用于Western Blots, recovery of some enzymatic activity, and some immunoprecipitation.注意：TRIzol LS试剂适用于液体样品（如，血液和病毒制品）。

TRIzol LS试剂与TRIzol试剂仅在它们成分的含量上不同，TRIzol LS试剂的浓度更高，因此相对于同个样品时，TRIzol LS试剂需要量更少。

TRIzol中文说明书

TRIzol2-8℃避光保存产品包装：100ml蓝色透明液体产品简介：TRIzol试剂适用于从细胞和组织中快速分离RNA。

TRIzol试剂有多组分分离作用,TRIzol使样品匀浆化，细胞裂解，溶解细胞内含物，同时保持RNA的完整性。

在加入氯仿离心后，溶液分为水相和有机相，RNA在水相中。

取出水相用异丙醇沉淀可回收RNA；用乙醇沉淀中间层可回收DNA；用异丙醇沉淀有机相可回收蛋白质。

TRIzol试剂可用于小量样品（50~100mg组织、5×106细胞）也适用于大量样品（≥1g组织、>107细胞）。

对人，动物，植物组织，细菌均适用，整个提取过程在一小时内即可完成。

分离的总RNA无蛋白质和DNA污染，可用于Northernblot，dotblot，ployA筛选，体外翻译，RNase 保护分析和分子克隆。

在用于RT-PCR时如果两条引物存在于一个单一外显子内，建议用无RNase的DNaseⅠ处理RNA样品，避免出现假阳性。

共纯化的DNA可用作标准，比较不同样品RNA的得率，也可用于PCR和酶切。

蛋白质可用于westernblotting。

注意事项：请勿直接接触皮肤或吞咽，以免灼伤。

如接触皮肤应立即用洗涤剂和大量水冲洗。

忌用乙醇擦洗，乙醇会加重灼伤。

预防RNase污染注意事项：1．经常更换新手套，皮肤上常带有的细菌，霉菌可能成为RNase的来源。

2．使用灭过菌的RNA专用塑料制品避免交叉污染。

3．RNA在TRIzol试剂中时不会被RNase污染，但提取后继续处理过程中应使用不含RNase 的塑料和玻璃器皿。

玻璃器皿可在150℃烘烤4小时，塑料制品可在0.5MNaOH中浸泡10分钟，然后用水彻底清洗，高压灭菌，即可去除RNase。

4．配制溶液应使用无RNase的水（将水加入处理过不含RNase的玻璃瓶中，加入DEPC至终浓度0.01℅v/v,放置过夜,高压灭菌。

注：DEPC有致癌之嫌，需小心操作。

invitrogen_Trizol提取中文说明书

TRIZOL® ReagentCat. No. 15596-026 Size: 100 mlStore at 2 to 8°C.警告：在与皮肤接触及吞咽有毒。

可导致烧伤。

与皮肤接触后，应立即用洗涤剂和大量水冲洗。

如感到身体不适，应就医（如需要，应出示本产品标签）。

本产品含有苯酚（108-95-2）和其他成分（NJTSRN 80100437-5000P）。

已经证明TRIZOL 在室温下可稳定保存12个月。

不过，我们建议在储存于2-8°C，以保证最佳性能。

描述：TRIzol试剂（美国专利号，5346994）是即用型细胞和组织总RNA提取试剂。

该试剂是一步法苯酚和异硫氰酸胍解决方案，是对Chomczynski和Sacchi开发的单步RNA提取法（1）的改善。

在匀质化或溶解样品中，TRIzol试剂可保持RNA的完整性，同时能破坏细胞及溶解细胞成分。

加入氯仿离心后，裂解液分离成水相和有机相。

RNA存在于水相。

水相转移后，RNA通过异丙醇沉淀回收。

移去水相后，样品中DNA和蛋白质可通过相继沉淀回收（2）。

用乙醇沉淀可从中间相得到DNA，加入异丙醇沉淀可从有机相得到蛋白质（2）。

与DNA的共纯化可能对不同样品得到的RNA的归一化有用。

此技术可完美应用于少量人类、动物、植物或细菌来源的组织（50-100毫克）和细胞（5×106），以及大量的组织（≥1 g）和细胞（>107）。

该TRIzol试剂方法简单，允许大量样本同时处理。

整个过程可在一小时内完成。

用TRIZOL提取总RNA可避免蛋白质和DNA 污染。

可用于Northern blot分析、斑点杂交、poly（A）+选择、体外翻译、RNA酶保护分析和分子克隆。

聚合酶链反应（PCR反应）中，当两条引物位于单个外显子时，推荐使用扩增级DNA酶I(Cat. No. 18068)处理分离出的RNA。

TRIzol试剂方便提取不同种类、不同分子大小的RNA。

Trizol原版说明书

TRI ZOL® ReagentCat. No. 15596-026 Size: 100 mlStore at 2 to 8°C.WARNING: Toxic in contact with skin and if swallowed. Causes burns. After contact with skin, wash immediately with plenty of detergent and water. If you feel unwell, seek medical advice (show label where possible). Phenol (108-95-2) and Other Components (NJTSRN 80100437-5000p).TRI ZOL has demonstrated stability of 12 months when stored at room temperature. However, we recommend storage at 2 to 8°C for optimal performance.Description:TRI ZOL Reagent (U.S.Patent No.5,346,994) is a ready-to-use reagent for the isolation of total RNA from cells and tissues. The reagent, a mono-phasic solution of phenol and guanidine isothiocyanate, is an improvement to the single-step RNA isolation method developed by Chomczynski and Sacchi (1). During sample homogenization or lysis, TRI ZOL Reagent maintains the integrity of the RNA, while disrupting cells and dissolving cell components. Addition of chloroform followed by centrifugation, separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase. After transfer of the aqueous phase, the RNA is recovered by precipitation with isopropyl alcohol. After removal of the aqueous phase, the DNA and proteins in the sample can be recovered by sequential precipitation (2). Precipitation with ethanol yields DNA from the interphase, and an additional precipitation with isopropyl alcohol yields proteins from the organic phase (2). Copurification of the DNA may be useful for normalizing RNA yields from sample to sample.This technique performs well with small quantities of tissue (50-100 mg) and cells (5 × 106), and large quantities of tissue (≥1 g) and cells (>107), of human, animal, plant, or bacterial origin. The simplicity of the TRI ZOL Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA isolated by TRI ZOL Reagent is free of protein and DNA contamination. It can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, and molecular cloning. For use in the polymerase chain reaction (PCR*), treatment of the isolated RNA with amplification grade DNase I (Cat. No. 18068) is recommended when the two primers lie within a single exon.TRI ZOL Reagent facilitates isolation of a variety of RNA species of large or small molecular size. For example, RNA isolated from rat liver, electrophoresed on an agarose gel, and stained with ethidium bromide, shows discrete bands of high molecular weight RNA between 7 kb and 15 kb in size, (composed of mRNA’s and hnRNA’s) two predominant ribosomal RNA bands at ~5 kb (28S) and at ~2 kb (18S), and low molecular weight RNA between 0.1 and 0.3 kb (tRNA, 5S). The isolated RNA has an A260/A280 ratio ≥1.8 when diluted into TE.Precautions for Preventing RNase Contamination:RNases can be introduced accidentally into the RNA preparation at any point in the isolation procedure through improper technique. Because RNase activity is difficult to inhibit, it is essential to prevent its introduction. The following guidelines should be observed when working with RNA. • Always wear disposable gloves. Skin often contains bacteria and molds that can contaminate an RNA preparation and be a source of RNases. Practice good microbiological technique to prevent microbial contamination.• Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment. For example, a laboratory that is using RNA probes will likely be using RNase A or T1 to reduce background on filters, and any nondisposable items (such as automatic pipettes) can be rich sources of RNases.• In the presence of TRI ZOL Reagent, RNA is protected from RNase contamination. Downstream sample handling requires that nondisposable glassware or plasticware be RNase-free. Glass items can be baked at 150°C for 4 hours, and plastic items can be soaked for 10 minutes in 0.5 M NaOH, rinsed thoroughly with water, and autoclaved.Other Precautions:• Use of disposable tubes made of clear polypropylene is recommended when working with less than 2-ml volumes of TRI ZOL Reagent.• For larger volumes, use glass (Corex) or polypropylene tubes, and test to be sure that the tubes can withstand 12,000 ×g with TRI ZOL Reagent and chloroform. Do not use tubes that leak or crack.• Carefully equilibrate the weights of the tubes prior to centrifugation.• Glass tubes must be sealed with parafilm topped with a layer of foil, and polypropylene tubes must be capped before centrifugation.INSTRUCTIONS FOR RNA ISOLATION:Caution: When working with TRI ZOL Reagent use gloves and eye protection (shield, safetyvapor.Unless otherwise stated, the procedure is carried out at 15 to 30°C, and reagents are at 15 to 30°C.Reagents required, but not supplied:• Chloroform• Isopropyl alcohol• 75% Ethanol (in DEPC-treated water)• RNase-free water or 0.5% SDS solution [To prepare RNase-free water, draw water into RNase-free glass bottles. Add diethylpyrocarbonate (DEPC) to 0.01% (v/v). Let stand overnight and autoclave. The SDS solution must be prepared using DEPC-treated, autoclaved water.] 1. HOMOGENIZATION (see notes 1-3)a. TissuesHomogenize tissue samples in 1 ml of TRI ZOL Reagent per 50-100 mg of tissue using aglass-Teflon® or power homogenizer (Polytron, or Tekmar's TISSUMIZER® orequivalent). The sample volume should not exceed 10% of the volume of TRI ZOLReagent used for homogenization.b. Cells Grown in MonolayerLyse cells directly in a culture dish by adding 1 ml of TRI ZOL Reagent to a 3.5 cmdiameter dish, and passing the cell lysate several times through a pipette. The amount ofTRI ZOL Reagent added is based on the area of the culture dish (1 ml per 10 cm2) and noton the number of cells present. An insufficient amount of TRI ZOL Reagent may result incontamination of the isolated RNA with DNA.c. Cells Grown in SuspensionPellet cells by centrifugation. Lyse cells in TRI ZOL Reagent by repetitive pipetting. Use1 ml of the reagent per 5-10 × 106 of animal, plant or yeast cells, or per 1 × 107 bacterialcells. Washing cells before addition of TRI ZOL Reagent should be avoided as thisincreases the possibility of mRNA degradation. Disruption of some yeast and bacterialcells may require the use of a homogenizer.OPTIONAL: An additional isolation step may be required for samples with high content of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous parts of plants. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12,000 ×g for 10 minutes at 2 to 8°C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer which should be removed. In each case, transfer the cleared homogenate solution to a fresh tube and proceed with chloroform addition and phase separation as described.2. PHASE SEPARATIONIncubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRI ZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 ×g for 15 minutes at 2 to 8°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRI ZOL Reagent used for homogenization.3. RNAPRECIPITATIONTransfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRI ZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 ×g for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.4. RNA WASHRemove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRI ZOL Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 ×g for 5 minutes at 2 to 8°C.5. REDISSOLVING THE RNAAt the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry for 5-10 minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/280 ratio < 1.6. Dissolve RNA in RNase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C. (Avoid SDS when RNA will be used in subsequent enzymatic reactions.) RNA can also be redissolved in 100% formamide (deionized) and stored at -70°C (5).RNA Isolation Notes:1. Isolation of RNA from small quantities of tissue (1 to 10 mg) or Cell (102 to 104) Samples:Add 800 µl of TRI ZOL to the tissue or cells. Following sample lysis, add chloroform and proceed with the phase separation as described in step 2. Prior to precipitating the RNA with isopropyl alcohol, add 5-10 µg RNase-free glycogen (Cat. No 10814) as carrier to the aqueous phase. To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to chloroform addition. The glycogen remains in the aqueous phase and is co-precipitated with the RNA. It does not inhibit first-strand synthesis at concentrations up to 4 mg/ml and does not inhibit PCR.2. After homogenization and before addition of chloroform, samples can be stored at -60 to -70°Cfor at least one month. The RNA precipitate (step 4, RNA WASH) can be stored in 75% ethanol at 2 to 8°C for at least one week, or at least one year at –5 to -20°C.3. Table-top centrifuges that can attain a maximum of 2,600 ×g are suitable for use in theseprotocols if the centrifugation time is increased to 30-60 minutes in steps 2 and 3.INSTRUCTIONS FOR DNA ISOLATION:After complete removal of the aqueous phase, as described in the RNA isolation protocol, the DNA in the interphase and phenol phase from the initial homogenate may be isolated. Following precipitation and a series of washes, the DNA is solubilized in 8 mM NaOH. Full recovery of DNA from tissues and culture cells permits the use of TRI ZOL Reagent for the determination of the DNA content in analyzed samples (2). Simultaneous extraction of genomic DNA allows for normalization of the results of Northern analysis per genomic DNA instead of the more variable total RNA or tissue weight. (Depending on the source, the DNA pellet obtained may require additional purification (e.g., phenol extraction) prior to other applications.Reagents required, but not supplied:• Ethanol• 0.1 M Sodium citrate in 10% ethanol• 75% Ethanol• 8 mM NaOHUnless otherwise stated, the procedure is carried out at 15 to 30°C.1. DNAPRECIPITATIONRemove the remaining aqueous phase overlying the interphase, and precipitate the DNA from the interphase and organic phase with ethanol. Add 0.3 ml of 100% ethanol per 1 ml of TRI ZOL Reagent used for the initial homogenization, and mix samples by inversion. Next, store the samples at 15 to 30°C for 2-3 minutes and sediment DNA by centrifugation at no more than 2,000 ×g for 5 minutes at 2 to 8°C.Careful removal of the aqueous phase is critical for the quality of the isolated DNA.2. DNAWASHRemove the phenol-ethanol supernatant, and if desired, save it for protein isolation. Wash the DNA pellet twice in a solution containing 0.1 M sodium citrate in 10% ethanol. Use 1 ml of the solution per 1 ml of TRI ZOL Reagent used for the initial homogenization. At each wash, store the DNA pellet in the washing solution for 30 minutes at 15 to 30°C (with periodic mixing) and centrifuge at 2,000 ×g for 5 minutes at 2 to 8°C. Following these two washes, suspend the DNA pellet in 75% ethanol (1.5-2 ml of 75% ethanol per 1 ml TRI ZOL Reagent), store for 10-20 minutes at 15 to 30°C (with periodic mixing) and centrifuge at 2,000 ×g for 5 minutes at 2 to 8°C.An additional wash in 0.1 M sodium citrate-10% ethanol solution is required for large pellets containing > 200 µg DNA or large amounts of a non-DNA material.3. REDISSOLVING THE DNAAir dry the DNA 5 to 15 minutes in an open tube. (DO NOT DRY UNDER CENTRIFUGATION; it will be more difficult to dissolve.) Dissolve DNA in 8 mM NaOH such that the concentration of DNA is 0.2 – 0.3 µg/µl. Typically add 300 – 600 µl of 8 mM NaOH to DNA isolated from 107 cells or 50 – 70 mg of tissue. Resuspending in weak base is HIGHLY recommended since isolated DNA does not resuspend well in water or in Tris buffers. The pH of the 8 mM NaOH is only ~9 and should be easily adjusted with TE or HEPES once the DNA is in solution. At this stage, the DNA preparations (especially from tissues) may contain insoluble gel-like material (fragments of membranes, etc.) Remove the insoluble material by centrifugation at >12,000 ×g for 10 minutes. Transfer the supernatant containing the DNA to a new tube. DNA solubilized in 8 mM NaOH can be stored overnight at 4°C; for prolonged storage, samples should be adjusted with HEPES to pH 7-8 (see table) and supplemented with 1 mM EDTA. Once the pH is adjusted, DNA can be stored at 4°C or –20°C.Quantitation and Expected Yields of DNAthe A260 of the resulting solution. Calculate the DNA content using the A260 value for double-stranded DNA. One A260 unit equals 50 µg of double-stranded DNA/ml. For calculation of cell number in analyzed samples, assume that the amount of DNA per 1 × 106 diploid cells of human, rat, and mouse origin equals: 7.1 µg, 6.5 µg, and 5.8 µg, respectively (3).Applications:Amplification of DNA by PCR:After redissolving the DNA in 8 mM NaOH, adjust the pH to 8.4 with 0.1 M HEPES (see table). Add 0.1 to 1.0 µg of the DNA sample to your PCR reaction mixture and perform the standard PCR protocol.Restriction endonuclease reactions:Adjust the pH of the DNA solution to a required value using HEPES (see table). Alternatively, samples may be dialyzed against 1 mM EDTA, pH 7 to pH 8.0. Use 3-5 units of enzyme per microgram of DNA. Use the conditions recommended by the manufacturer for the particular enzyme, and allow the reaction to proceed for 3 to 24 h. In a typical assay, 80-90% of the DNA is digestible.pH Adjustment of DNA Samples Dissolved in 8 mM NaOH:(For 1 ml of 8 mM NaOH use the following amounts of 0.1 M or 1 M HEPES, free acid.)Final pH 0.1 M HEPES (µl)Final pH 1 M HEPES (µl)8.4 86 7.2 238.2 93 7.0 328.0 1017.8 1177.5 159DNA Isolation Notes:1. The phenol phase and interphase can be stored at 2 to 8°C overnight.2. Samples suspended in 75% ethanol can be stored at 2 to 8°C for months.3. Samples dissolved in 8 mM NaOH can be stored overnight at 2 to 8°C. For long-term storage,adjust the pH to 7-8, and adjust the EDTA concentration to 1 mM.INSTRUCTIONS FOR PROTEIN ISOLATION:Proteins are isolated from the phenol-ethanol supernatant obtained after precipitation of DNA with ethanol (step 1, DNA PRECIPITATION). The resulting preparation can be analyzed for the presence of specific proteins by Western blotting (2).Reagents required, but not supplied:• Isopropyl alcohol• 0.3 M Guanidine hydrochloride in 95% ethanol• Ethanol• 1% SDS1. PROTEINPRECIPITATIONPrecipitate proteins from the phenol-ethanol supernatant (approximate volume 0.8 ml per 1 ml of TRI ZOL Reagent) with isopropyl alcohol. Add 1.5 ml of isopropanol per 1 ml of TRI ZOL Reagent used for the initial homogenization. Store samples for 10 minutes at 15 to 30°C, and sediment the protein precipitate at 12,000 ×g for 10 minutes at 2 to 8°C.2. PROTEINWASHRemove the supernatant and wash the protein pellet 3 times in a solution containing 0.3 M guanidine hydrochloride in 95% ethanol. Add 2 ml of wash solution per1 ml of TRI ZOL Reagent used for the initial homogenization. During each wash cycle, store the protein pellet in the wash solution for 20 minutes at 15 to 30°C and centrifuge at 7,500 ×g for 5 minutes at 2 to 8°C. After the final wash, vortex the protein pellet in 2 ml of ethanol. Store the protein pellet in ethanol for 20 minutes at 15 to 30°C and centrifuge at 7,500 ×g for 5 minutes at 2 to 8°C.3. REDISSOLVING THE PROTEIN PELLETVacuum dry the protein pellet for 5-10 minutes. Dissolve it in 1% SDS by pipetting. Complete dissolution of the protein pellet may require incubating the sample at 50°C. Sediment any insoluble material by centrifugation at 10,000 ×g for 10 minutes at 2 to 8°C, and transfer the supernatant to a fresh tube. The sample is ready for use in Western blotting or may be stored at -5 to -20°C for future use. Protein Isolation Notes:1. The protein pellet suspended in 0.3 M guanidine hydrochloride-95% ethanol or in ethanol canbe stored for at least one month at 2 to 8°C, or for at least one year at -5 to -20°C.2. The following protocol is an alternative approach that allows for more efficient recovery ofproteins. Dialyze the phenol-ethanol supernatant against three changes of 0.1% SDS at 2 to 8°C. Centrifuge the dialyzed material at 10,000 ×g for 10 minutes. Use the clear supernatant for Western blotting.3. Proteins may be quantified by the Bradford method as long as the concentration of SDS is lowenough (<0.1%) so that it will not interfere. Methods that do not have detergent-interface problems, and that do not rely on A260/A280 measurements may be used (traces of phenol may cause overestimation of protein concentrations).Troubleshooting Guide:RNA ISOLATION•Expected yields of RNA per mg of tissue or 1 × 106 cultured cellsLiver and spleen, 6-10 µgKidney,3-4µgSkeletal muscles and brain, 1-1.5 µgPlacenta,1-4µgEpithelial cells (1 × 106 cultured cells), 8-15 µgFibroblasts,(1× 106 cultured cells) 5-7 µg•Low yieldIncomplete homogenization or lysis of samples.Final RNA pellet incompletely redissolved.•A260/A280 ratio <1.65RNA sample was diluted in water instead of TE prior to spectrophotometric analysis. Low ionic strength and low pH solutions increase absorbance at 280 nm (6,7).Sample homogenized in too small a reagent volume.Following homogenization, samples were not stored at room temperature for 5 minutes.The aqueous phase was contaminated with the phenol phase.Incomplete dissolution of the final RNA pellet.•RNA degradationTissues were not immediately processed or frozen after removal from the animal.Samples used for isolation, or the isolated RNA preparations were stored at -5 to-20°C, instead of -60 to -70°C.Cells were dispersed by trypsin digestion.Aqueous solutions or tubes were not RNase-free.Formaldehyde used for agarose-gel electrophoresis had a pH below 3.5.•DNA contaminationSample homogenized in too small a reagent volume.Samples used for the isolation contained organic solvents (e.g., ethanol, DMSO), strong buffers, or alkaline solution.• Proteoglycan and polysaccharide contaminationThe following modification of the RNA precipitation (step 3) removes these contaminating compounds from the isolated RNA. Add to the aqueous phase 0.25 ml of isopropanol followed by 0.25 ml of a high salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl) per 1 ml of TRI ZOL Reagent used for the homogenization. Mix the resulting solution, centrifuge and proceed with the isolation as described in the protocol. The modified precipitation effectively precipitates RNA while maintaining polysaccharides and proteoglycans in a soluble form. A combination of the modified precipitation with an additional centrifugation of the initial homogenate (note 2, RNA isolation protocol) is required to isolate pure RNA from plant material containing a very high level of polysaccharides.DNA ISOLATION•Expected yields of DNA per mg of tissue or 1 × 106 cultured cellsLiver and kidney, 3-4 µgSkeletal muscles, brain, and placenta 2-3 µgCultured human, rat, and mouse cells (1 × 106), 5-7 µgFibroblasts,5-7µg•Low yieldIncomplete homogenization or lysis of samples.Final DNA pellet incompletely redissolved.•A260/280 ratio <1.70DNA sample was diluted in water instead of TE prior to spectrophotometric analysis.Phenol was not sufficiently removed from the DNA preparation. Wash the DNA pellet anadditional time with 0.1 M sodium citrate in 10% ethanol.•DNA degradationTissues were not immediately processed or frozen after removal from the animal.Samples used for isolation, or the isolated RNA preparations were stored at -5 to-20°C, instead of -60 to -70°C.Samples were homogenized with a Polytron or other high speed homogenizer.•RNA contaminationIncomplete removal of aqueous phase.DNA pellet insufficiently washed with 0.1 M sodium citrate in 10% ethanol.•Other applicationsPrior to use in PCR amplification, adjust the pH to 8.4.For digestion of the DNA with restriction endonucleases, adjust the pH to the desired value, use 3-5 units of enzyme per µg of DNA, and allow the reaction to go for 3-24 hours underoptimal conditions for the particular enzyme. Typically 80-90% of the DNA is digested. PROTEIN ISOLATION•Low yieldIncomplete homogenization or lysis of samples.Final DNA pellet incompletely redissolved.•Protein degradationTissues were not immediately processed or frozen after removing from the animal.•Band deformation in PAGEProtein pellet insufficiently washed.References:1. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156.2. Chomczynski, P. (1993) Biotechniques 15, 532.3. Ausubel, F.M., et.al, eds. (1990) Current Protocols in Molecular Biology, Vol.2, GreenePublishing Assoc. and Wiley-Interscience, New York, p.A.1.5.4. Simms, D., Cizdziel, P.E., Chomczynski, P.(1993) F OCUS®15, 99.5. Bracete, A.M., Fox, D.K., and Simms, D. (1998) F OCUS 20, 82).6. Wilfinger, W., Mackey, K. and Chomczynski, P. (1997) BioTechniques 22, 474.7. Fox, D.K. (1998) F OCUS 20, 37.Teflon® is a registered trademark of E. I. Du Pont de Nemours & Co.TISSUMIZER® is a registered trademark of Tekmar Co.TRI ZOL ® is a registered trademark of Molecular Research Center, Inc.*PCR is covered by a patent held by Hoffman LaRoche Corporation.Cat No. 15596-026。

invitrogen trizol rna抽提说明书

RNA抽提全过程(TRIZOL)一，准备工作1，实验器具与材料：（1）移液枪：1ml、200ul、10ul（2）吸头：1ml、200ul、20ul（3）吸头台：放置1ml吸头的一个，放置200ul和20ul的吸头一个（4）EP管1.5ml、100ul（5）玻璃研磨器（6）容量瓶：1000ml（7）盐水瓶：100ml（8）15ml塑料管一个（配75％乙醇用）2，实验器具的处理与准备（1）塑料制品：（包括吸头、EP管等）将塑料制品逐个浸泡于1‰DEPC水中（必要时小枪头需要用吸管打入DEPC水）37℃过夜，然后送至高压3次，后在80℃烘烤箱中烘干（或置于37℃中8小时左右烘干），试验前将枪头放入吸头台。

或直接购买经DEPC处理的枪头和EP管,每盒大约30元,基本上试剂公司都可以购买。

（2）玻璃制品：（主要是玻璃研磨器）先泡酸过夜，冲洗干净后，在1‰DEPC水中泡8小时左右，37℃烘干，用蒙锡纸包裹送至干烤3次（或180度干烤）。

（3）金属制品：（镊子等）先洗干净，再送干烤3次。

（不需要泡DEPC水）3，试剂配制和准备：（1）DEPC水：泡实验器具的DEPC水的配制：1000ml双蒸水中加1mlDEPC，放在1000ml 容量瓶中静置4小时后备用。

配75％乙醇的DEPC水的配制：100ml盐水瓶内装40ml双蒸水，加40ulDEPC，37℃过夜，送至高压。

（2）75％乙醇（要在抽提时现配）：用无水乙醇和DEPC水配制（DEPC水:无水乙醇＝1:3），然后放于-20℃备用。

（3）异丙醇：放入棕色瓶（4）氯仿：放入棕色瓶（5）Trizol：100ml/瓶存放于4℃二，抽提时注意事项：全程佩戴一次性手套和口罩，手套要勤换，避免戴上的一次性的手套接触可疑污染物。

三，抽提步骤1．匀浆化作用取约100mg鼠脑组织放于玻璃研磨器内，先加0.2ml的Trizol溶液，研磨组织后，倒入1.5mlEP管中，再在研磨器内加入0.8ml的Trizol溶液洗，后全部再倒入EP管中。

trizol 使用说明书中文版

Trizol使用说明书一、分离纯化的基本原理研究基因的表达和调控时常常要从组织和细胞中分离和纯化RNA。

RNA质量的高低常常影响cDNA库，RT-PCR和Northern Blot等分子生物学实验的成败。

Trizol是一种新型总RNA抽提试剂，内含异硫氰酸胍等物质，能迅速破碎细胞，抑制细胞释放出的核酸酶。

二、用户需自备的试剂和材料无水乙醇、氯仿、Glycogen（可能需要）、 1.5ml Eppendorf管（RNase-free）、 Tips （RNase-free）三、准备工作RNase酶非常稳定，是导致RNA降解最主要的物质。

它在一些极端的条件可以暂时失活，但限制因素去除后有迅速复性。

用常规的高温高压蒸气灭菌方法和蛋白抑制剂都不能是RNase完全失活。

它广泛存在于人的皮肤上，因此，在与RNA制备有关的分子生物学实验时，必须戴手套。

RNase的又一污染源是取液器，根据取液器制造商的要求对取液器进行处理。

一般情况下采用用DEPC配制的70%乙醇擦洗取液器的内部和外部，基本达到要求。

取RNase-free的物品时必须戴手套。

1、料制品的处理尽可能使用无菌，一次性塑料制品，已标明RNase-Free 的塑料制品，如没有开封使用过通常没有必要再次处理。

对于国产塑料制品，原则上都必须处理方可使用。

处理步骤如下：1）在玻璃烧杯中注入去离子水，加入DEPC使DEPC的终浓度为0.1%。

注意：DEPC为剧毒物，活性很强，小心在通风柜中使用。

2）处理的塑料制品放入一个可以高温灭菌的容器中，注入DEPC水溶液，使塑料制品的所有部分都浸泡到溶液中。

3）在通风柜中室温处理过夜。

4）将DEPC水溶液小心倒到废液瓶中，用铝箔封住含已DEPC水处理过的塑料制品的烧杯，高温高压蒸气灭菌至少30分钟。

5）烘箱用合适的温度烘拷至干燥。

置于干净处备用。

2、璃玻和金属物品250°C烘烤3小时以上。

四、从组织中提取总RNA1)液氮研磨，组织块直接放入研钵中，加入少量液氮，迅速研磨，待组织变软，再加少量液氮，再研磨，如此三次，按50-100mg组织/ml Trizol加入Trizol，转入离心管进行第2步操作。

invitrogentrizol试剂盒的说明书

invitrogentrizol试剂盒的说明书从组织中提取总RNA1)液氮研磨，组织块直接放入研体中，加入少量液氮，迅速研磨，待组织变软，再加少量液氮，再研磨，如此三次，按50-100mg组织/mlTrizol加入Trizol，转移入离心管进行第2步操作。

2) 匀浆：组织样品按50-100mg/mlTrizol 加入Trizol，另外，组织体积不能超过Trizol 体积的10%，否则匀浆效果会不好，用电动匀浆器充分匀浆约需1-2分钟。

从细胞中提取总RNA1) 培养贴壁细胞：不须消化，可直接用Trizol进行消化、裂解，Trizol体积按10cm2/ml 比例加入。

2) 悬浮细胞可直接收集、裂解，每1ml Trizol可裂解5×106动物、植物或酵母细胞，或107细菌细胞。

2．细胞或组织加Trizol后，室温放置5min，使其充分裂解。

注：此时可放入-70℃长期保持。

3．12000rpm 离心5min，弃沉淀。

4．按200ul氯仿/ml Trizol加入氯仿，振荡混匀15分钟，室温放置15min。

注：禁用漩涡振荡器，以免基因组DNA断裂。

5．4℃12000g离心15min。

6．吸取上层水相，至另一离心管中。

注：1）千万不要吸取中间界面。

2)若同时提取DNA和蛋白质，则保留下层酚相存于4℃冰箱,若只提RNA，则弃下层酚相。

7．按0.5ml异丙醇/ml Trizol 加入异丙醇混匀，室温放置5-10min。

8．4℃ 12000g离心10min，弃上清，RNA沉于管底。

9．按1ml 75%乙醇/ml Trizol加入75%乙醇，温和振荡离心管，悬浮沉淀。

10．4℃ 8000g离心5min，尽量弃上清。

11．室温晾干或真空干燥5-10min。

注：RNA样品不要过于干燥，否则很难溶解。

12．可用50ul H2O，TE buffer或0.5%SDS溶解RNA样品，55-60℃5-10min。

注：H2O、TE或0.5%SDS均须用DEPC处理并高压。

Trizol说明书

Trizol说明书产品简介：本公司生产的Trizol试剂适用于从细胞或组织中分离总RNA，结果产量高、完整性好、纯度高，省时、省力。

提取的总RNA可用于cDNA合成、RT-PCR、Northern Blot、Dot blot、Primer Extension、Poly A RNA筛选、体外翻译或其他基因工程用途。

该试剂能促进不同种属不同分子量大小的多种RNA的析出。

例如，从大鼠肝脏抽提（mRNA 的RNA琼脂糖凝胶电泳时，可见许多介于7kb和15kb之间不连续的高分子量条带，和hnRNA成分）两条优势核糖体RNA条带位于~5 kb (28S)和~2 kb (18S)，28S与18S电泳条带亮度比值约为2：1，低分子量RNA介于0.1 和 0.3 kb之间 (tRNA, 5S)。

当抽提的RNA用TE稀释时其A260/A280比值≥1.8。

TRizol试剂保存于4℃可达12个月。

下图从K562细胞总提取的总RNA电泳效果图下表从组织和细胞中提取的总RNA量组织或细胞（mg、1×106 ）总RNA产量（ug）肝、脾8~20肾3~5骨骼肌、脑组织1~5胎盘1~5上皮细胞8~20纤维母细胞5~20试剂盒组成产品编号131904 131905 保存条件Trizol 50ml 100ml 4℃说明书 1 份操作者提供及注意事项1、新的氯仿、异丙醇、70%酒精（0.1% DEPC-H2O配制）、4℃离心机（速度约12000rpm）2、1.5ml离心管，1ml、200ul、20ul的吸头，需经过0.1% DEPC-H2O 37℃处理过夜，高压消毒烤干待用；研磨器用水洗干净后，需要180℃高温干烤3小时。

3、在抽提RNA过程中任一环节的不正确操作都可能导致RNA酶的污染。

由于RNA 酶的活性很难完全抑制，预防其污染十分必要。

全程佩戴一次性手套、口罩。

皮肤经常带有细菌和霉菌，可能污染RNA的抽提并成为RNA酶的来源。

新景生物 Trizol 试剂说明书

邮编：310030电话：传真：Trizol 试剂说明书产品组成Trizol 试剂规格Cat.No.5301100Cat.No.5301005说明书100ml 5.5ml 1份产品储存与有效期请将产品储存于2~8℃，有效期为3年。

技术支持杭州新景生物试剂开发有限公司研发部：电话：400-0099-857，QQ:869912443，微信公众号：simgenbio ，e-mail:。

产品介绍Trizol 试剂是即用型细胞和组织总RNA 提取试剂。

在匀质化或溶解的样品中，Trizol 试剂可保持RNA 的完整性，同时能破坏细胞及溶解细胞成分。

加入Buffer EX 或氯仿离心后，样本溶解物分离成水相和有机相，RNA 存在于水相中，DNA 和蛋白质处于有机相及相间。

水相中的RNA 可通过异丙醇沉淀回收；如果有需要，样品中DNA 和蛋白质可通过相继沉淀再次回收。

Trizol 试剂提取的总RNA 可用于Northern blot 分析、斑点杂交、poly （A ）+选择、体外翻译、RNA 酶保护分析和分子克隆。

Trizol 试剂可除去样本中大部分DNA ，但不能彻底去除DNA ，因此在RT-PCR 反应中，如果设计的两条引物位于单个外显子中时，应选用DNase I （Simgen Cat.No.8003050）处理分离出的RNA ，或者选择含有DNA 酶消化步骤的cDNA 第一链合成试剂盒（Simgen Cat.No.7306100）合成cDNA 。

用户需自备的试剂与物品1.Buffer EX （Simgen Cat.9025100）或氯仿、异丙醇2.RNase-free 水或者DEPC 处理水3.75％乙醇（用DEPC 处理水配制）4.RNase-free 的1.5ml 离心管和移液器及吸头5.一次性手套及防护用品和纸巾6.台式小量离心机（可配离心1.5ml 离心管和2ml 离心管的转子）Trizol 试剂操作视频7.可能需要液氮与研钵，可能需要18-25号针头的注射器（动物组织）8.可能需要PBS 溶液、无水乙醇、0.1M 柠檬酸钠（溶于10%乙醇）、8mM NaOH （DNA提取）9.可能需要5ml 离心管、0.3M 盐酸胍（溶于95%乙醇）、1%SDS （蛋白质提取）使用前准备1.扫描右侧二维码了解Trizol 试剂操作步骤，了解“防止RNA 酶污染的注意事项”。

TRIzol中文说明书

T R I z o l中文说明书(共5页) -本页仅作为预览文档封面，使用时请删除本页-TRIzol2-8℃避光保存产品包装：100ml蓝色透明液体产品简介：TRIzol试剂适用于从细胞和组织中快速分离RNA。

TRIzol试剂有多组分分离作用,TRIzol使样品匀浆化，细胞裂解，溶解细胞内含物，同时保持RNA的完整性。

在加入氯仿离心后，溶液分为水相和有机相，RNA在水相中。

取出水相用异丙醇沉淀可回收RNA；用乙醇沉淀中间层可回收DNA；用异丙醇沉淀有机相可回收蛋白质。

TRIzol试剂可用于小量样品（50~100mg组织、5×106细胞）也适用于大量样品（≥1g组织、>107细胞）。

对人，动物，植物组织，细菌均适用，整个提取过程在一小时内即可完成。

分离的总RNA无蛋白质和DNA污染，可用于Northernblot，dotblot，ployA筛选，体外翻译，RNase保护分析和分子克隆。

在用于RT-PCR时如果两条引物存在于一个单一外显子内，建议用无RNase 的DNaseⅠ处理RNA样品，避免出现假阳性。

共纯化的DNA可用作标准，比较不同样品RNA的得率，也可用于PCR和酶切。

蛋白质可用于westernblotting。

注意事项：请勿直接接触皮肤或吞咽，以免灼伤。

如接触皮肤应立即用洗涤剂和大量水冲洗。

忌用乙醇擦洗，乙醇会加重灼伤。

预防RNase污染注意事项：1．经常更换新手套，皮肤上常带有的细菌，霉菌可能成为RNase的来源。

2．使用灭过菌的RNA专用塑料制品避免交叉污染。

3．RNA在TRIzol试剂中时不会被RNase污染，但提取后继续处理过程中应使用不含RNase的塑料和玻璃器皿。

玻璃器皿可在150℃烘烤4小时，塑料制品可在中浸泡10分钟，然后用水彻底清洗，高压灭菌，即可去除RNase。

4．配制溶液应使用无RNase的水（将水加入处理过不含RNase的玻璃瓶中，加入DEPC至终浓度℅v/v,放置过夜,高压灭菌。

Trizol试剂

Trizol Reagent/总RNA提取试剂发布日期：[2007-7-13] 共阅[7177]次Trizol Reagent/总RNA提取试剂说明书产品简介TRIzol是从细胞和组织中提取总RNA的即用型试剂，在样品裂解或匀浆过程中，TRIzol能保持RNA完整性。

加入氯仿后，溶液分为水相和有机相，RNA在水相中。

取出水相，用异丙醇可沉淀回收RNA。

TRIzol试剂可用于小量样品（50－100mg组织、5×106细胞），也可用于大量样品（≥1g组织或≥107个细胞），对人、动物、植物和细菌、血液提取都适用，可同时处理大量不同样品。

一个小时内即可完成反应，提取的总RNA没有DNA和蛋白质污染，可用于Northern blot、RT-PCR、Dot blot、RNase保护分析等。

本试剂为红颜色，便于区分水相和有机相，同时最大限度的保持RNA的稳定性。

保存条件2-8℃避光保存12个月。

实验前需要准备的试剂氯仿、异丙醇、75%乙醇（用RNase-free水配制）、RNase-free的水。

操作步骤1. 匀浆处理（Homogenization）组织中提取总RNA1) 植物组织：植物叶片直接放入研钵中，加入少量液氮，迅速研磨成粉末，每50-100mg植物叶片加入1ml TRIzol。

2) 动物组织：按10-30mg组织加入1ml TRIzol，用电动匀浆器或者一次性研磨杵充分匀浆。

培养细胞中提取总RNA1) 贴壁细胞：无须胰酶消化，可直接用TRIzol进行裂解，每10平方厘米培养面积加1ml TRIzol。

2) 悬浮细胞：可直接离心收集、裂解，每1ml TRIzol可裂解5×106动物或酵母细胞，或107细菌细胞。

血液中提取总RNA直接取新鲜的血液，加入3倍体积红细胞裂解液，混匀后室温放置10分钟，10,000 rpm离心1分钟。

彻底吸弃上清，收集白细胞沉淀。

每100-200μl血液收集的白细胞沉淀加入1ml TRIzol。