invitrogen trizol试剂盒的说明书

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。

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.。

Invitrogen合成生物学服务产品手册说明书

Invitrogen 合成生物学服务产品手册•高品质引物和探针合成生物学服务合成生物学是什么？合成生物学是分子生物学与系统生物学的组合，使用工程学的原则来设计生物系统和生物工厂，其目的是创造出改进的生物功能以应对当前与未来的挑战。

我们相信合成生物学将会改变我们获取能源、生产食物以及优化工业过程的方式，并能检测、预防与治愈疾病。

我们致力于为科研人员提供卓越的技术和解决方案。

通过科学与工程，这一独特的领域能让科研人员研究、修改、创造和再创造高度复杂的生化途径、DNA 序列、基因以及自然生物系统，以便能够理解并解答一些关于生命最有挑战性的问题。

其中Pleasanton, California 工厂拥有：• ISO 9001认证• ISO 13485认证• GMP 认证我们在全球有9个生产基地：• Pleasanton, California • Regensburg, Germany • Aukland, New Zealand • Haneda, Japan • Inchinnan, UK • San Paulo, Brazil • Suzhou, China • Beijing, China • Guangzhou, China立足中国在中国，我们有苏州、北京、广州三地工厂进行合成生物学服务，服务内容包括引物合成和探针合成。

苏州、北京和广州工厂均获得ISO 9001认证，其中苏州工厂另有ISO 14000认证。

我们有多样化的仪器：• 25+台Jurassics 合成仪• 3台Applied Biosystems 3900合成仪• 3台Mermade12高通量合成仪• 1台AKTA 大规格合成仪• 5台LCMS 质检仪器• 1台 Agilent 96CE PRO II • 15+台 HPLC 纯化仪满足不同类型的应用需求：• PCR • qPCR • STR • SSR • NGSInvitrogen 引物合成服务已经超过了20年，我们在20余年的专业DNA 合成中积累了丰富的经验和良好的业内声誉。

诺维赞trizol说明书

诺维赞trizol说明书1、该法从细胞中提取RNA——用匀浆器将组织磨碎，加入TRIzol处理组织。

5分钟后加入氯仿，以每分钟12000转离心5分钟，样品即分成水样层、中间层和有机层。

2、RNA存在于水样层中，收集水样层后可以通过异丙醇沉淀RNA来还原。

3、在除去水样层后，中间层中的DNA和蛋白质也能相继以沉淀的方式还原：乙醇能使中间层的DNA沉淀析出，在中间层中加入异丙醇能沉淀出蛋白质。

4、每100mlTRIzol可以抽提100个六孔板中的样品或100个50-80mg的组织样品。

无论样品是人、动物、植物还是细菌组织，该方法对少量的组织（50-100mg)和细胞（约五百万个）及大量的组织（≥1g）和细胞（超过一千万个）均有较好的分离效果。

5、每一百万细胞用TRIzol抽提可得5-15微克RNA，每毫克组织用TRIzol抽提可得1-10微克RNA（产量因细胞和组织不同而异）。

6、TRIzol操作上的简便性允许其同时处理多个样品，所有的操作可以在一小时内完成。

7、TRIzol抽提的总RNA能够避免DNA和蛋白质的污染，故而能够作RNA印迹分析、斑点杂交、poly(A)+选择、体外翻译、RNA 酶保护分析和单分子克隆（PCR）。

8、如果是用于PCR，当两条引物位于单一外显子内时，建议用级联扩大的DNaseI(Cat.No.18068）来处理抽提的总RNA。

9、TRIzol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，从大鼠肝脏抽提的RNA经过琼脂糖凝胶电泳并用溴化乙啶染色，可见许多介于7kb和15kb之间的不连续的高分子量条带（mRNA和hnRNA成分），两条优势核糖体RNA条带位于~5kb（28S）和~2kb（18S），低分子量RNA介于0.1和0.3kb之间(tRNA,5S）。

当抽提的RNA用TE稀释时，其A260/A280比值≥1.8。

抽提小RNA时宜-70℃沉淀过夜。

trizol说明书

TRIzol™ ReagentCatalog Numbers 15596026 and 15596018Doc. Part No. 15596026.PPS Pub. No. MAN0001271 Rev.A.0WARNING! Read the Safety Data Sheets (SDSs) and follow thehandling instructions. Wear appropriate protective eyewear,clothing, and gloves. Safety Data Sheets (SDSs) are availablefrom /support.Product informationInvitrogen™ TRIzol™ 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 of phenol, 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 RNase activity while disrupting cells and dissolving cell components during sample homogenization. TRIzol™ Reagent allows for simultaneous processing of a large number of samples, and is an improvement to the single-step RNA isolation method developed by Chomcynski and Sacchi (Chomczynski and Sacchi, 1987).TRIzol™ Reagent allows 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.TRIzol™ Reagent can also be used with Phasemaker™ Tubes to isolate RNA. Refer to TRIzol™ Reagent and Phasemaker™ Tubes Complete System User Guide (MAN0016163) for the full protocol.Contents and storageRequired materials not suppliedUnless otherwise indicated, all materials are available through . MLS: Fisher Scientific () or other major laboratory supplier.Table 1 Materials required for RNA, DNA, and protein isolationTable 2 Materials required for RNA isolationTable 3 Materials required for DNA isolationTable 4Materials required for protein isolationInput sample requirementsIMPORTANT! Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.Fresh tissues or tissues stored in RNA™ Stabilization Solution (Cat.No. AM7020).Procedural guidelines•Perform all steps at room temperature (20–25°C) unless otherwise noted.•Use cold TRIzol™ Reagent if the starting material contains high levels of RNase, such as spleen or pancreas samples.•Use disposable, individually wrapped, sterile plastic ware andsterile, disposable RNase-free pipettes,pipette tips, and tubes.•Wear disposable gloves while handling reagents and RNAsamples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocolprogresses from crude extracts to more purified materials.•Always use proper microbiological aseptic techniques whenworking with RNA.•Use RNase Zap™ RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.Lyse samples and separate phases1.Lyse and homogenize samples in TRIzol™ Reagent according toyour starting material.•Tissues:Add 1 mL of TRIzol™ Reagent per 50–100 mg of tissue to thesample and homogenize using a homogenizer.•Cell grown in monolayer:a.Remove growth media.b.Add 0.3–0.4 mL of TRIzol™ Reagent per 1 × 105—107 cellsdirectly to the culture dish to lyse the cells.c.Pipet the lysate up and down several times to homogenize.•Cells grown in suspension:a.Pellet the cells by centrifugation and discard thesupernatant.b.Add 0.75 mL of TRIzol™ Reagent per 0.25 mL of sample (5–10 × 106 cells from animal, plant, or yeasty origin or 1 ×107cells of bacterial origin) to the pellet.Note: Do not wash cells before addition of TRIzol™ Reagentto avoid mRNA degradation.c.Pipet the lysate up and down several times to homogenize.Note: The sample volume should not exceed 10% of the volume of TRIzol™ Reagent used for lysis.STOPPING POINT Samples can be stored at 4°C overnight or at –20°C for up to a year.2.(Optional) If samples have a high fat content, centrifuge the lysatefor 5 minutes at 12,000 × g at 4–10°C, then transfer the clearsupernatant to a new tube.3.Incubate for 5 minutes to permit complete dissociation of thenucleoproteins complex.4.Add 0.2 mL of chloroform per 1 mL of TRIzol™ Reagent used forlysis, then securely cap the tube.5.Incubate for 2–3 minutes.6.Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.The mixture separates into a lower red phenol-chloroform, andinterphase, and a colorless upper aqueous phase.7.Transfer the aqueous phase containing the RNA to a new tube.8.Transfer the aqueous phase containing the RNA to a new tube byangling the tube at 45° and pipetting the solution out.IMPORTANT! Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase. Proceed directly to “Isolate RNA“ on page 2.Save the interphase and organic phase if you want to isolate DNA or protein. See “Isolate DNA“ on page 3 or “Isolate proteins“ onpage 4 for detailed procedures. The organic phase can be stored at 4°C overnight.Isolate RNAa.(Optional) If the starting sample is small (<106 cells or <10 mg of tissue), add 5–10 µg of RNase-freeglycogen as a carrier to the aqueous phase.Note: The glycogen is co-precipitated with the RNA, but does not interfere with subsequentapplications.b.Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzol™ Reagent used for lysis.c.Incubate for 10 minutes.d.Centrifuge for 10 minutes at 12,000 × g at 4°C.Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.e.Discard the supernatant with a micropipettor.1Precipitate the RNAa.Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis.Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C.b.Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.c.Discard the supernatant with a micropipettor.d.Vacuum or air dry the RNA pellet for 5–10 minutes.IMPORTANT! Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure totalsolubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.2Wash the RNA3Solubilize the RNAa.Resuspend the pellet in 20–50 µL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipettingup and down.IMPORTANT! Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymaticreactions.b.Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.Proceed to downstream applications, or store the RNA at –70°C.4Determine the RNA yieldTable 5 Typical RNA (A260/280 of >1.8) yields from various starting materialsIsolate DNA from the interphase and the lower phenol-chloroform phase saved from “Lyse samples and separate phases“ on page 2.1Precipitate the DNAa.Remove any remaining aqueous phase overlying the interphase.This is critical for the quality of the isolated DNA.b.Add 0.3 mL of 100% ethanol per 1 mL of TRIzol™ Reagent used for lysis.c.Cap the tube, mix by inverting the tube several times.d.Incubate for 2–3 minutes.e.Centrifuge for 5 minutes at 2000 × g at 4°C to pellet the DNA.f.Transfer the phenol-ethanol supernatant to a new tube.The supernatant is used for protein isolation (see “Isolate proteins“ on page 40, if needed, and can bestored at –70°C for several months.2Wash the DNAa.Resuspend the pellet in 1 mL of 0.1 M sodium citrate in 10% ethanol, pH 8.5, per 1 mL of TRIzol™Reagent used for lysis.b.Incubate for 30 minutes, mixing occasionally by gentle inversion.Note: The DNA can be stored in sodium citrate/ethanol for at least 2 hours.c.Centrifuge for 5 minutes at 2000 × g at 4°C.d.Discard the supernatant with a micropipettor.e.Repeat step 2a–step 2d once.Note: Repeat step 2a–step 2d twice for large DNA pellets (>200 µg).f.Resuspend the pellet in 1.5–2 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis.g.Incubate for 10–20 minutes, mixing occasionally by gentle inversion.Note: The DNA can be stored in 75% ethanol at several months at 4°C.h.Centrifuge for 5 minutes at 2000 × g at 4°C.i.Discard the supernatant with a micropipettor.j.Vacuum or air dry the DNA pellet for 5–10 minutes.IMPORTANT! Do not dry the pellet by vacuum centrifuge.3Solubilize the DNAa.Resuspend the pellet in 0.3–0.6 mL of 8 mM NaOH by pipetting up and down.Note: We recommend resuspending the DNA is a mild base because isolated DNA does not resuspendwell in water or Tris buffer.b.Centrifuge for 10 minutes at 12,000 × g at 4°C to remove insoluble materials.c.Transfer the supernatant to a new tube, then adjust pH as needed with HEPES.Proceed to downstream applications, or store the DNA at 4°C overnight. For longer-term storage at –20°C,adjust the pH to 7–8 with HEPES and add 1 mM EDTA.4Determine the DNA yieldTable 6 Typical DNA (A260/280 of 1.6–1.8) yields from various starting materialsIsolate the proteins from the phenol-ethanol supernatant saved from “Precipitate the DNA“ on page 3 using either “Precipitate the proteins“ on page 4 or “Dialyse the proteins“ on page 5.1Precipitate the proteinsa.Add 1.5 mL of isopropanol to the phenol-ethanol supernatant per 1 mL of TRIzol™ Reagent used forlysis.b.Incubate for 10 minutes.c.Centrifuge for 10 minutes at 12,000 × g at 4°C to pellet the proteins.d.Discard the supernatant with a micropipettor.2Wash the proteinsa.Prepare a wash solution consisting of 0.3 M guanidine hydrochloride in 95% ethanol.b.Resuspend the pellet in 2 mL of wash solution per 1 mL of TRIzol™ Reagent used for lysis.c.Incubate for 20 minutes.Note: The proteins can be stored in wash solution for at least 1 month at 4°C or for at least 1 year at –20°C.d.Centrifuge for 5 minutes at 7500 × g at 4°C.e.Discard the supernatant with a micropipettor.f.Repeat step 2b–step 2e twice.g.Add 2 mL of 100% ethanol, then mix by vortexing briefly.h.Incubate for 20 minutes.i.Centrifuge for 5 minutes at 7500 × g at 4°C.j.Discard the supernatant with a micropipettor.k.Air dry the protein pellet for 5–10 minutes.IMPORTANT! Do not dry the pellet by vacuum centrifuge.3Solubilize the proteinsa.Resuspend the pellet in 200 µL of 1% SDS by pipetting up and down.Note: To ensure complete resuspension of the pellet, we recommend that you incubate the sample at50°C in a water bath or heat block.b.Centrifuge for 10 minutes at 10,000 × g at 4°C to remove insoluble materials.c.Transfer the supernatant to a new tube.Proceed directly to downstream applications, or store the sample at –20°C.•Measure protein concentration by Bradford assay.Note: SDS concentration mush be <0.1%.4Determine the protein yieldDialyse the proteins1.Load the phenol-ethanol supernatant into the dialysis membrane.Note: The phenol-ethanol solution can dissolve some types ofdialysis membranes (cellulose ester, for example). Test dialysistubing with 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 change 4hours later (at 20 hours), and the final change 2 hours later (at 22 hours).Note: A SDS concentration of at least 0.1% is required toresolubilize the proteins from the pellet. If desired, the SDS can be diluted after solubilization.3.Centrifuge the dialysate for 10 minutes at 10,000 × g at 4°C.4.Transfer the supernatant containing the proteins to a new tube.5.(Optional) Solubilize the pellet by adding 100 µL of 1% SDS and100 µL of 8 M urea.Proceed directly to downstream applications, or store the sample at –20°C.TroubleshootingLimited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at /support.ReferencesChomczynski, 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., and Ried, T. 2007 Isolation and solubilization of proteins after TRIzol® extraction of RNA and DNA from patient material following prolonged storage. BioTechniques 42, 467-472The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Revision history: Pub. No. MAN00001271Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.For support visit /support or email techsupport@。

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用法

TRIZOL（invitrogen life techologies）一．RNA提取1.-80o C取标本2.取组织3.液氮中陶瓷研钵研磨，组织呈粉状时转移到预先称重的5毫升的塑料离心管中，离心管需要DEPC水处理后，灭菌。

称重，得组织100mg4.加入TRIZOL 1.5ml（-60 o C—-70o C下，可存放1个月）5.室温保温5min6.加入氯仿0.3ml，盖紧手震动混匀15s7.室温2-3min8.4 o C，12000g 15min9.取上清约0.8ml，转移到1.5ml离心管中，管子处理同上，去净上清的中下层液体用于DNA和蛋白质的提取10.加异丙醇0.7ml，混匀，室温下10min11.4 o C，10000g 10min12.弃上清，加1.4ml 75%乙醇（以DEPC处理灭菌水配制）混匀13.4 o C，7000g 5min14.弃上清，室温干燥5min15.DEPC处理灭菌纯水100μl溶解，-70 o C保存二．DNA提取1.去净上清的中下层液体约0.6ml，加入100%乙醇0.45ml，混匀2.室温下2-3min3.4 o C，5000g 5min4.去除液体，可保留用为蛋白提取5.加入0.1M柠檬酸钠75%乙醇溶液洗涤1.4ml DNA沉淀，室温下30分钟，间断混匀6.4 o C，2000g 5min7.重复洗涤一次8.加入0.1M柠檬酸钠75%乙醇溶液洗涤1.4ml DNA沉淀，室温下15分钟，间断混匀9.4 o C，2000g 5min10.室温下干燥5-15min11.在300-600μl 8mM NaOH中溶解，DNA浓度约为0.2-0.3 μg/ μl12.若有不溶物，12000g 10min13.上清可以在4 o C存放过夜14.长时间保存需要用HEPES加1mM EDTA调整pH至7-8。

三．蛋白质提取1.转移上清到5ml的离心管中，大约体积为1ml2.加入2.3ml异丙醇（1.5ml/mlTRIZOL），室温下保温10min3.4 o C，12000g 10min4.加入3ml（2ml/mlTRIZOL）含0.3M盐酸胍的95%乙醇，室温下20min5.4 o C，7500g 5min6.重复洗涤3次7.加入2ml乙醇，室温下20min8.4 o C，7500g 5min9.真空干燥蛋白沉淀5－10min，1%SDS溶解10.完全溶解需要在50 o C保温，不溶物可4 o C 10000g 10min离心去除11.上清转移至新管，可用于Western blotting或－20o C存放。

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中文说明书

T R I z o l中文说明书-CAL-FENGHAI.-(YICAI)-Company One1TRIzol2-8℃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。

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。

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 LS 试剂使用中文说明书

TRIZOL LS 试剂使用中文说明书TRIzol??LS?试试(ambion);中文试明试,试试格室试存号温10296?010?????????????????100ml10296?028200ml试品描述TRIzol?LS试试适用于液试品～如人、试植物、酵母、试菌或者病毒源的液试品体来体～提取高试量的试RNA;DNA和蛋白试也可以,全试程只需1小试。

TRIzol?LS试试里含有、硫试酸和其成分～由于在试试品试程中能有效地抑制酚异胍它匀很RNase活性～所以TRIzol?LS试试能试持RNA完整性。

TRIzol?LS试试允试同试试理大量的试品。

TRIzol?LS试试能一试品里有序分提取从个离RNA、DNA 和蛋白试。

TRIzol?LS试试均试化试品后～加入试～能看试分试试象～上试是透明的含仿RNA的水相～中试试～下试是试色的有机相;含有DNA和蛋白试,。

水相RNA 用丙醇能淀分～中试试或异沉离者下试中的DNA用乙醇淀分～丙醇淀能使蛋白试沉离异沉从酚?醇相的上液中清沉来沉淀出。

淀出的RNA、DNA和蛋白试洗试试～重试后用于下游。

脱分的离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分的离DNA可以用于PCR, and Restriction Enzyme digestion, and Southern Blots.分的蛋白试可以用于离Western Blots, recovery of some enzymatic activity, and someimmunoprecipitation.注意,TRIzol?LS试试适用于液试品;如～血液和病毒制品,。

翻译好的罗氏公司Tunel试剂盒操作说明书 (2)

罗氏(R o c h e)公司T u n e l试剂盒操作说明书(Insitucelldeathdetectionkit-POD法)一、原理：TUNEL（TdT-mediateddUTPnickendlabeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。

其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdTEnzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radishperoxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）EnzymeSolution酶溶液：TdT10×、2号（紫盖）LabelSolution标记液：荧光素标记的dUTP1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5μl20×DAB+1μL30%H2O2+94μlPBS）、ProteinaseK工作液（10-20μg/mlin10mMTris/HCl，pH7.4-8）或细胞通透液（0.1%TritonX-100溶于0.1%柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase1（3000U/ml–3U/mlin50mMTris-HCl，pH7.5，10mMMgCl2，1mg/mlBSA）等。

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

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_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 Invitrogen提取RNA和DNA介绍

Trizol Invitrogen提取RNA和DNA介绍TRIzol提取RNA和DNA简要说明准备试剂：TRIzol氯仿100%异丙醇%乙醇无RNase的水handy pestle操作步骤：RNA操作在冰上进行1、每50～100mg组织加入1ml TRIzol ，handy pestle匀浆RNALater保存的组织，（其实组织50mg提出的RNA大大富余）2、将匀浆样品在室温（15～30℃）放置5分钟，使核酸蛋白复合物完全分离。

3、每使用1ml TRIzol加入0.2ml氯仿，用手剧烈振荡15秒，室温放置3分钟。

4、4℃12,000×g离心15分钟。

样品分为三层：底层为黄色有机相，上层为无色水相和一个中间层。

RNA主要在水相中，水相体积约为所用TRIzol试剂的约50%。

5、用200ul的枪2枪把水相转移到新管中（样本足够因此少抽以防止DNA污染）。

6、1mlTRIzol加入0.5ml100%异丙醇，颠倒混匀，室温放置10分钟。

7、4℃12,000×g离心10分钟，移去上清，之后可以看到RNA 沉淀。

8、1ml TRIzol至少加1ml 80%乙醇。

（-20°放一年） 10、vortex混匀，4℃下7500×g离心5分钟，弃上清，也可见沉淀。

9、室温放置干燥RNA沉淀，大约5～10分钟即可.加入无RNase 的水（乳腺癌100mg样本加50ul浓度大约为2ug/ul），用枪头吸打几次，稍微离心，55～60℃放置10分钟使RNA溶解。

可-80℃保存一段时间，最好马上逆转录。

10、260比280是1.8-2.1（低可能是污染，也可能测时候的问题）产量公式：260×稀释倍数×40=ug/ml DNA的分离准备试剂：乙醇0.1M柠檬酸钠（含10%乙醇） 75%乙醇8mM NaOH 操作步骤：样品加氯仿分层后，移去上层水相， 1mlTRIzol加0.3ml无水乙醇混匀，颠倒混匀，室温放置3分钟 4℃2000×g离心5分钟。

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