TransZol法rna提取试剂盒说明
RNAzol RT RNA 提取剂说明书

Product codeE01010AProduct nameRNAzol® RT RNA Isolation ReagentContact manufacturerGeneCopoeia, Inc.9620 Medical Center Drive, Suite 101 Rockville, MD 20850 USAPhone: 301-762-0888 Toll free: 1-866-360-9531 Fax: 301-762-3888 Chemical Name CAS-No Weight %Phenol 108-95-2 30-60 Guanidine isothiocyanate 593-84-0 15-40 Ammonium thiocyanate 1762-95-4 7-13Contact with acids or bleach liberates toxic gases. DO NOT ADD acids or bleach to any liquid wastes containing this product. We recommend handling all chemicals with caution. GHS – ClassificationSignal Word DANGERHealth HazardsMaterial Safety Data SheetRevision Date: September 12, 2014Health Hazards (continued)Physical hazardsNot hazardousHazard StatementsH314 - Causes severe skin burns and eye damageH341 - Suspected of causing genetic defectsH373 - May cause damage to organs through prolonged or repeated exposureH412 - Harmful to aquatic life with long lasting effectsH302 - Harmful if swallowedH312 - Harmful in contact with skinH332 - Harmful if inhaledH335 - May cause respiratory irritationPrecautionary StatementsP301 + P312 - IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwellP304 + P340 - IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathingP301 + P330 + P331 - IF SWALLOWED: rinse mouth. Do NOT induce vomitingP303 + P361 + P353 - IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin withwater/showerP305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsingP310 - Immediately call a POISON CENTER or doctor/physicianP308 + P313 - IF exposed or concerned: Get medical advice/attentionP273 - Avoid release to the environmentP280 - Wear protective gloves/protective clothing/eye protection/face protectionPrinciple Routes of ExposurePotential Health EffectsEyes Causes burns. Risk of serious damage to eyes. Corrosive to the eyesand may cause severe damage including blindness.Skin Causes burns. Possible risk of irreversible effects. Harmful in contactwith skin.Irritating to skin and mucous membranes.Inhalation Harmful by inhalation.Ingestion Harmful if swallowed. Ingestion causes burns of the upper digestive andrespiratory tracts. Ingestion may cause gastrointestinal irritation, nausea,vomiting and diarrhea.Specific effectsCarcinogenic effects Phenol has been classified by the International Agency for Research onCancer (IARC) as not classifiable as to carcinogenicity to humans(Group 3).Mutagenic effects Not ApplicableReproductive toxicity Not ApplicableSensitization Not ApplicableTarget Organ Effects SkinLungsLiverSpleenKidneyHMISSkin contactWash off immediately with soap and plenty of water while removing all contaminated clothes and shoes. Call a physician immediately.Eye contactIF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Rinseimmediately with plenty of water, also under the eyelids, for at least 15 minutes. Call a physician immediately.Ingestion Call a physician or poison control center immediately. Rinse mouth. Do not induce vomiting without medical advice. Never give anything by mouth to an unconscious person.InhalationRemove to fresh air. Call a physician or poison control center immediately.Notes to physicianTreat symptomatically.Suitable extinguishing media Dry chemical. Carbon dioxide (CO2). Water spray. Foam. Special protective equipment for firefighters Wear self-contained breathing apparatus and protective suit.Australia HazChem Code 2XPersonal precautionsELIMINATE all ignition sources (no smoking, flares, sparks or flames in immediate area). Use personal protection equipment. Avoid contact with skin, eyes or clothing. Ensure adequate ventilation. Keep people away from and upwind of spill/leak. Evacuate personnel to safe areas.Methods for cleaning upPrevent product from entering drains. Soak up with inert absorbentmaterial. Neutralize spill with slaked lime, sodium bicarbonate or crushed limestone. Collect powdered material and deposit in sealed containers and dispose of phenol as hazardous waste. Isolate area and deny entry.Environmental precautionsPrevent further leakage or spillage if safe to do so. Prevent product from entering drains. Do not allow material to contaminate ground water system.See Section 12 for more informationHandling Always wear recommended Personal Protective Equipment. Avoidcontact with skin, eyes or clothing. Remove all sources of ignition. Storage Keep containers tightly closed in a cool, well-ventilated place. Keep awayfrom heat, sparks, flame and other sources of ignition (i.e., pilot lights,electric motors and static electricity). Protect from sunlight.Exposure limitsEngineering measures Use in a chemical fume hoodPersonal protective equipment Personal Protective Equipment requirements are dependent on the userinstitution's risk assessment and are specific to the risk assessment foreach laboratory where this material may be used.Respiratory protection In case of insufficient ventilation wear suitable respiratory equipment Respirator Up to 50 ppmRecommendations, NationalInstitute of OccupationalSafety and Health, U.S.(APF = 10) Any air-purifying half-mask respirator with organic vaporcartridge(s) in combination with an N95, R95, or P95 filter. The followingfilters may also be used:N99, R99, P99, N100, R100, P100.(APF = 10) Any supplied-air respiratorUp to 125 ppm:(APF = 25) Any supplied-air respirator operated in a continuous-flowmode.(APF = 25) Any powered air-purifying respirator with an organic vaporcartridge in combination with a high-efficiency particulate filter.Up to 250 ppm:(APF = 50) Any air-purifying full-facepiece respirator equipped withorganic vapor cartridge(s) in combination with an N100, R100, or P100filter.(APF = 50) Any air-purifying, full-facepiece respirator (gas mask) with achin-style, front- or back-mounted organic vapor canister having anN100, R100, or P100 filter.(APF = 50) Any powered, air-purifying respirator with a tight-fittingfacepiece and organic vapor cartridge(s) in combination with a high-efficiency particulate filter.(APF = 50) Any self-contained breathing apparatus with a full facepiece.(APF = 50) Any supplied-air respirator with a full facepiece.Emergency or planned entry into unknown concentrations or IDLHconditions:(APF = 10,000) Any self-contained breathing apparatus that has a fullfacepiece and is operated in a pressure-demand or other positive-pressure mode.(APF = 10,000) Any supplied-air respirator that has a full facepiece andis operated in a pressure-demand or other positive-pressure mode incombination with an auxiliary self-contained positive-pressure breathingapparatus.Escape:(APF = 50) Any air-purifying, full-facepiece respirator (gas mask) with achin-style,front- or back-mounted organic vapor canister having an N100, R100, orP100 filter. /Any appropriate escape-type, self-contained breathingapparatus.Hand protection Impervious gloves. S24 - Avoid contact with skin. S36 - Wear suitableprotective clothing.Eye protection Tight sealing safety goggles.Skin and body protection Impervious clothing.Hygiene measures Contaminated work clothing should not be allowed out of the workplace.Avoid contact with skin, eyes or clothing. Handle in accordance withgood industrial hygiene and safety practice.Environmental exposureControls Prevent product from entering drains.General informationForm Liquid.Appearance Red, maroon.Odor Medicinal, sweet, tar-like.Boiling point/range°C No data available °F No data availableMelting point/range°C No data available °F No data availableFlash point °C No data available °F No data availableAutoignition temperature°C No data available °F No data availableOxidizing properties No information availableWater solubility SolubleStability Stable under normal conditions.Materials to avoid Strong oxidizing agents. Strong acids. Isocyanates. Heat. Nitriles,Nitrides. Alkaline earth metals. Strong oxidizers, alkali metals andalkaline earth metals may cause fires or explosions.Hazardous decomposition Toxic gas. Sulphur oxides. Hydrogen cyanide (hydrocyanic acid). Carbonoxides,Products Nitrogen Oxides.Polymerization Hazardous polymerization does not occur.Acute toxicityPrinciple Routes of ExposurePotential Health EffectsEyes Causes burns. Risk of serious damage to eyes Corrosive to the eyes andmay cause severe damage including blindness.Skin Causes burns. Possible risk of irreversible effects Harmful in contact withskin. Irritating to skin and mucous membranes.Inhalation Harmful by inhalationIngestion Harmful if swallowed Ingestion causes burns of the upper digestive andrespiratory tracts Ingestion may cause gastrointestinal irritation, nausea,vomiting and diarrheaCarcinogenic effects Phenol has been classified by the International Agency for Research onCancer (IARC) as not classifiable as to carcinogenicity to humans(Group 3).Mutagenic effects No information available.Reproductive toxicity No information available.Sensitization No information available.Target organ effects Skin.Lungs.Liver.Spleen.Kidney.Ecotoxicity Harmful to aquatic organisms, may cause long-term adverse effects inthe aquatic environment.Chronic aquatic toxicity Category 3Mobility See log PowBiodegradation Inherently biodegradableBioaccumulation No information availableDispose of contents/containers in accordance with local regulations.IATAProper shipping name Corrosive liquid, n.o.s. (guanidine thiocyanate-phenol solution).Hazard Class8Subsidiary class NonePacking group IIUN-No1760ERG Code153US Federal RegulationsSARA 313This product contains the following toxic chemical(s) subject to the notification requirements of section 313 of Title III of the Superfund Amendments and Reauthorization Act of 1986. This law requires certain manufacturers to report on annual emissions of specified chemicals and chemical categories. Please note that if you repackage, or otherwise redistribute, this product to industrial customers, a notice similar to this one should be sent to those customers:Clean Air Act, Section 112 Hazardous Air Pollutants (HAPs) (see 40 CFR 61)This product contains the following HAPs:US state regulationsCalifornia Proposition 65This product does not contain any Proposition 65 chemicals.WHMIS Hazard ClassD1A - Very toxic materialsE - Corrosive materialThis product has been classified in accordance with the hazard criteria of the Controlled Products Regulations (CPR) and the MSDS contains all the information required by the CPR.Reason for revision Not Applicable. SDS sections updated.For research use only."The above information was acquired by diligent search and/or investigation and the recommendations are based on prudent application of professional judgment. The information shall not be taken as being all inclusive and is to be used only as a guide. All materials and mixtures may present unknown hazards and should be used withcaution. Since the Company cannot control the actual methods, volumes, or conditions of use, the Company shall not be held liable for any damages or losses resulting from the handling or from contact with the product as described herein.THE INFORMATION IN THIS SDS DOES NOT CONSTITUTE A WARRENTY, EXPRESSED OR IMPLIED, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PUPOSE"End of Safety Data Sheet。
康为RNA提取试剂盒使用说明书

RNA试剂盒所含药品:Component CW0559S 50 preps DNase I(DNA酶Ⅰ)1000 U 10×Reaction Buffer(10×缓冲液)1000 μlBuffer RL 35 mlBuffer RLC 35 mlBuffer RW1 40 mlBuffer RW2(concentrate)11 ml RNase-Free Water(无RNA酶的水)10 ml50Spin Columns FL with Collection Tubes(含收集管的吸附柱FL)50Spin Columns RM with Collection Tubes(含收集管的吸附柱RM)50RNase-Free Centrifuge Tubes(1.5 ml)(无RNA酶的离心管)自备试剂:β-巯基乙醇、无水乙醇(新开封或提取RNA专用)。
实验前准备及重要注意事项:1.预防RNase污染,应注意以下几方面:1)使用无RNase的塑料制品和枪头,避免交叉污染。
2)配制溶液应使用无RNase的水。
3)操作人员戴一次性口罩和手套,实验过程中要勤换手套。
2.预防RNase污染,应注意以下几方面:1)使用无RNase的塑料制品和枪头,避免交叉污染。
2)玻璃器皿应在使用前于180℃高温下干烤4小时,塑料器皿可在0.5 M N aOH 中浸泡10分钟,用水彻底冲洗后高压灭菌。
3)配制溶液应使用无RNase的水。
4)操作人员戴一次性口罩和手套,实验过程中要勤换手套。
3.提取的样品避免反复冻融,否则影响RNA提取的量和质量。
4.Buffer RL在使用前请加入β-巯基乙醇,1 ml Buffer RL加10 μl β-巯基乙醇。
加入β-巯基乙醇的Buffer RL室温可保存1个月。
Buffer RLC使用时不需加β-巯基乙醇。
5.第一次使用前应按照试剂瓶标签的说明先在Buffer RW2中加入无水乙醇。
天漠科技 总RNA提取试剂盒说明书

总RNA提取试剂盒(配合TRIZOL使用)Catalog No. TR205-50 (50次反应含DNaseI)TR205-200 (200次反应含DNaseI)TR205-50N (50次反应无DNaseI)TR205-200N (200次反应无DNaseI)Highlights∙适用于从细胞,组织,酵母菌,植物,细菌或生物液体(任何TRIZOL或类似产品可以裂解的样品)中提取到总RNA(含microRNA)。
∙无需进行相分离,无需使用氯仿,无需沉淀过程。
∙提取到的RNA不含DNA,并可应用于Q-PCR,高通量测序等实验。
∙整个过程仅需10分钟。
Ver.1.0.9产品组成:50200RNA洗涤液1 室温40 ml 160 ml第一次使用前按说明加指定量乙醇RNA洗涤液2室温12 ml 48 ml第一次使用前按说明加指定量乙醇DNase I -20℃ 300μl x 1管(5U/μl) 300μl x 4管(5U/μl)DNA消化液室温 4 ml 16 mlRNase-free H2O 室温 6 ml 30 ml2号柱室温50个200个收集管(2ml)室温50个200个特性:1.样品来源:TRIZOL等类似试剂裂解的样品。
2.样品范围:≥17核苷酸。
3.RNA纯度:高质量的RNA可应用于高通量测序等敏感的下游实验。
4.RNA结合量:每个柱子可最多结合50μg的RNA。
试剂制备RNA洗涤液在使用之前一定要配好,添加好乙醇后在试剂瓶上做好标记!!!添加10ml或40ml的无水乙醇(95-100%)到40ml或160ml的RNA洗涤液1中。
添加48ml100%的乙醇(或52 ml95%的乙醇)到12ml的RNA洗涤液2添加192ml100%的乙醇(或208 ml95%的乙醇)到48ml的RNA洗涤液2操作步骤:整个操作步骤是由2个步骤组成:(I)样品裂解匀浆(I I)样品纯化(I)样品裂解匀浆此步骤主要参考TRIZOL等类似裂解试剂说明书的裂解液用量,以下给出我公司提供的可完美替换TRIZOL的TRIcom试剂用量作为参考。
总RNA提取试剂盒操作流程

总RNA提取试剂盒操作流程试剂配制:DNA酶1:将550ul无核酶水加入DNA酶1瓶中,分装32瓶,每瓶,可用3次。
注意,轻轻混匀,不要震荡。
置于-20度保存,避免反复冻融,不宜超过3次以上。
RNA裂解液:将1-硫代甘油按2%(V/V)加入RNA 裂解液中,可在4度保存6个月。
RNA洗液:将70ml无水乙醇加入到40mlRNA洗液中。
步骤:1、贴壁细胞裂解物的制备。
胰酶消化离心后得细胞悬浮液,取*103-5*106 细胞,离心后收集细胞沉淀。
(不同样品最适起始用量和裂解液、稀释液的使用量。
悬浮或贴壁细胞*103-5*106使用量,裂解液300ul,稀释液300ul。
)2、加入300ul的RNA裂解液,吹打混匀,移至管中二RNA提取1、加入300ul的RNA稀释液,用移液枪混匀,室温放置3-5分钟,(如果样品比较珍贵,可以选择裂解物加入稀释液后70℃下加热3分钟,可以提高RNA的得率)2、以最大速度离心5分钟,吸取上清液置于另一管中3、加入倍上层清液体积的无水乙醇,移液器吹打3-4次,混匀,4、取出离心柱/细心管(离心柱已安放在收集管上),将混合物移至离心柱中,如果混合物体积过大,可分2次上样过柱。
*g离心1分钟,弃掉滤液5、加入600ulRNA洗液,*g离心45秒,弃掉滤液6、配置DNA酶1孵育液,DNA酶1缓冲液5ul+dna酶1 液5ul+无核酶水40ul.注意轻轻混匀,不要震荡。
(这是提取一管RNA需要的量)7、加入50ulDNA酶1孵育液到吸附膜中央,室温放置15分钟8、加入600ulRNA 洗液,*g离心45秒,弃掉滤液。
将离心柱重新安置于收集管上,*g离心2分钟9、将离心柱转移到洗脱管上,在离心柱膜中央加入50-200ul无核酶水,室温下静置2分钟,*g离心1分钟。
将RNA保存在-70℃。
10、温馨提示,如果将第一次洗脱下来的洗脱液重新加回到离心柱中央,室温下静置2分钟,*g离心1分钟,再次洗脱RNA,可以提高RNA的得率。
试剂盒提取RNA

RNA提取(试剂盒法)1、取适量的植物组织(0.5g)多次加液氮研磨至粉末状,将粉末状样品(50-100mg)加入到含有450uL Buffer RL(使用前加入50×DTT Solution,1mL Buffer RL中加入20uL50×DTT,现用现配)的1.5mL灭菌管中,移液器反复吸打至无明显沉淀2、12,000rpm,5min,4℃3、上清转移到新的1.5mL管4、加入1∕2倍体积的无水乙醇(可能出现沉淀)吸打混匀5、立即将混合液(含沉淀)转入RNA Spin Column(含2mLCollection Tube)中。
(若混合液体积大于600uL,分批加入,每次加入体积不大于600uL)6、12,000rpm,1min,弃滤液,将RNA Spin Column放回2mLCollection Tube7、将500uL Buffer RW A加入RNA Spin Column,12,000rpm,30s,弃滤液8、将600uL Buffer RWB加入RNA Spin Column,12,000rpm,30s,弃滤液(RWB使用前加70mL100%乙醇)(沿RNA Spin Column管壁四周加入Buffer RWB有助于完全冲洗粘附于管壁上的盐分)9、DNase I消化⑴DNase I反应液:5uL 10 × DNase I Buffer,4uL Recombinant DNase I(RNase free,5U∕uL),41uL RNase free dH2O到新的1.5ml RNase free Tube 混匀⑵向RNA Spin Column膜中央加入50uLDNase I 室温静置15min⑶向RNA Spin Column膜中央加入350uL Buffer RWB,12,000rpm,30s,弃滤液10、重复步骤811、将RNA Spin Column重新置于2mLCollection Tube,12,000rpm,2min12、将RNA Spin Column置于1.5mL RNase free Collection Tube上,在RNA Spin Column 膜中央加入50-200uL RNase free dH2O或0.1%DEPC水,室温静置5min13、12,000rpm,2min 洗脱RNA14、若想提高RNA收量可再向膜中央加入50-200uL RNase free dH2O或0.1%DEPC水;若想提高RNA浓度可将第一次的洗脱液重新加回至RNA Spin Column室温静置5min,12,000rpm,2min 洗脱RNA。
病毒基因组RNA提取试剂盒使用说明书

病毒RNA 提取试剂盒Virus RNA Kit(目录号:HS0408)产品包装自备试剂无水乙醇储存条件蛋白酶K 于-20℃,其他组分室温(15 ~ 25℃)产品简介本试剂盒适用于从新鲜或冷冻的血浆、血清和无细胞体液中提取高质量的病毒RNA 。
无需使用苯酚、氯仿等有机溶剂抽提,独特的缓冲液/蛋白酶K 体系能迅速裂解病毒,使病毒蛋白与RNA 分离,在蛋白酶K 的作用下降解病毒蛋白,在高盐状态下将病毒RNA 选择性吸附于硅基质膜上,再通过快速的漂洗、离心步骤,去除蛋白等杂质,最后低盐的洗脱缓冲液将高纯度的病毒RNA 从吸附柱膜上洗脱下来。
本试剂盒操作简单、快速,所得病毒RNA 不含蛋白、核酸酶和其他杂质,可直接用于RT-PCR 、Real-Time PCR 、印迹等分子生物学实验。
产品特点1.简便快速,1小时内可获得高纯度的病毒RNA 。
2.无需有机溶剂抽提,使用安全。
北京厚生博泰科技有限公司 Beijing Hooseen Biotech Co., Ltd.3.重复性好,产量高。
4.所得病毒RNA纯度高,无污染物和抑制剂,方便下游应用。
注意事项1.血清或血浆避免反复冻融,否则会使蛋白变性或产生沉淀,导致提取的RNA片段小,提取量下降。
2.如缓冲液Buffer GB、Buffer RD结晶或产生沉淀,可在56℃水浴溶解。
3.所有离心步骤均为室温下操作。
4.本试剂盒也可提取高质量的病毒DNA,操作步骤相同。
操作步骤1. 取1.5 ml离心管(自备),加入20 ul的Proteinase K溶液。
2. 向离心管中加入200 ul血清或血浆,然后再加入200 ul Buffer GB,涡旋震荡15 sec。
(注意:1、样本体积不足200 ul可以加入0.9% NaCl(自备)补足。
2、为确保样本有效裂解,加入Buffer GB后,需将样本与Buffer GB充分混匀。
)3.56℃孵育15 min,短暂离心,将管壁上的溶液收集到管底。
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总RNA抽提试剂盒

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)、 ips (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。
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TransZol法RNA提取步骤
准备试剂:氯仿、无水乙醇。
1.菌体处理
(1)将发酵后的菌体小心地转入1.5ml离心管中,用纯水冲洗菌体,12000rpm离心2min,仔细去除培养基上清。
(2)加入TransZol TM Up (每≤2×109个细菌中加入1ml TransZol TM Up)。
(3)用移液枪反复吸吹直至裂解液中无明显沉淀。
(4)室温静置5min。
2. 每使用1ml TransZol TM Up,加入0.2ml氯仿或50ul 4-Bromoanisole(间溴茴香醚),剧烈震荡30s,室温孵育3min。
3. 10000×g 4℃离心15min。
此时溶液分成三层,无色的水相(上层)、中间层,粉红色有机相(下层)。
RNA分布在水相中,水相体积约为所用TransZol TM Up 试剂的50%-60%(为了避免吸到中间层导致DNA污染,可以适当留下一部分水相)。
4. 转移无色的水相于新的RNase-free离心管中,加入等体积的无水乙醇(此时可能会出现沉淀),轻轻颠倒混匀。
5. 将得到的溶液和沉淀一起加入离心柱中,12,000×g 室温离心30s,弃掉流出液(如果体积大于离心柱容量,可以分几次完成)。
6. 加入500ul CB9,12,000×g 室温离心30s,弃掉流出液。
7. 重复步骤6一次。
8. 加入500ul WB9(使用前请先检查是否加入无水乙醇),12,000×g 室温离心30s,弃掉流出液。
9. 重复步骤8一次。
10.12,000×g 室温离心2min,彻底去除残留的乙醇,在室温静置数分钟彻底晾干离心柱。
11.将离心柱放入RNase-free Tube(试剂盒已配)中,加50-200ul RNase-free Water 在离心管的中央,室温静置1min。
12. 12,000×g 室温离心1min,洗脱RNA。
(可选步骤:为获得更多的RNA,建议重复步骤11和12进行二次洗脱)。
13.将RNA置于-80℃保存。