OMEGArna提取试剂盒中文说明

OMEGA小提试剂盒提取质粒步骤（中文翻译版）

质粒提取（小提，mini kit）1、将携带目的质粒的大肠杆菌接种于含10~15ul基础培养基/氨苄西林培养介质的50ml培养瓶中。

2、室温下5000×g离心10min。

3、弃去上清，剩余沉淀物中加入500ul的SolutionⅠ/RNase A，彻底混匀。

4、将混悬液移至新的2ml离心管中，加入500ul SolutionⅡ，轻柔彻底混匀，可得清亮的细菌裂解物，室温下孵育2min（混匀时用力过大，可破碎出染色体DNA，使目的质粒纯度下降）。

5、向4中液体加入250ul预冷的Buffer N3，轻柔、彻底混匀，直到出现白色絮状沉淀，4℃≥12000×g离心10min（可室温，最好4℃）（Buffer应彻底混匀，若混合物粘稠呈棕色或呈球状，应多混匀几次以中和溶液，溶液的彻底中和对于获得好的产出是必要的）。

6、小心吸取并将上清液转移进新的1.5ml离心管1：0.1的比例向上清液中加入ETR Solution 混匀溶液并于冰上孵育10min，孵育过程中颠倒几次以混匀（加入ETR Solution后，细菌裂解物将出现浑浊，但冰上孵育后将变澄清）（勿用2ml离心管收集上清，因为2ml离心管中有太多液体时，ETR Solution将悬浮于溶液中）。

7、将6中液体于42℃孵育5min，溶液将再次变浑。

室温下12000×g离心3min，ETR Solution将于离心管底部形成蓝色层。

8、将上层水相转移入新的2ml离心管中，按1：0.5的比例加入无水乙醇（室温，96~100％），轻柔混匀，室温下孵育1~2min。

9、将8中的溶液取700ul到柱子中，组装收集管，室温下1000×g 离心1min，弃去收集管中通过柱子的液体，柱子和收集管重复利用。

10、重复9中步骤，直到收集的细菌裂解物全部用完。

11、将500ul Buffer HB加入柱子中，室温下10000×g离心1min，弃去收集管中废液（目的：将残存的蛋白污染物除去，是获得高质量DNA所必需的）。

质粒中提中文版(Omega)

中提质粒步骤材料准备：灭菌250或500mL三角瓶，托盘天平，5mL移液枪及枪头，卫生纸，废液缸，记号笔，50mL 圆底离心管，15mL尖底离心管步骤：1.集菌将100mL菌液分批倒入50mL圆底离心管中，室温12000rpm离心1min，弃流出液体，打开盖倒置吸水纸上控干。

2.加入2.5mL solution I（提前加入RNase，4℃保存），重悬细菌沉淀（可使用5mL移液枪吹打）。

3.加入2.5mL solution II，轻柔混匀（颠倒离心管7-10次），以获得清亮的裂解物，室温静置3-5min。

（此步主要是碱裂解细菌，使其中的蛋白质和DNA变性，反应时间不能太长，否则容易使大肠杆菌的基因组DNA断裂成小片段，污染质粒DNA）4.加入1.25mL Buffer N3，轻柔混匀（颠倒离心管7-10次），直至形成白色沉淀，室温静置2-3min。

5.4℃12000rpm离心10min，使白色沉淀沉于底部。

6.取出一个Lysate Clearance Filter Syringe，抽出活塞，小心将5中液体全部转移至注射器中（注射器可不放活塞直接直立于4中的离心管中，液体不会出来），室温静置2min。

7.将6中注射器放置于一新的15mL离心管，轻推注射器活塞，使液体流入离心管中。

8.加入0.1倍体积的ETR（约600uL）至过滤后的液体中，混匀（颠倒离心管7-10次），冰浴20min，期间颠倒离心管几次。

（此期间准备一个Hind-Bind DNA midi Column加入2mL GPS Buffer，室温静置10min，4000rpm离心5min）9.42℃温浴5min，溶菌液变浑浊，室温4000rpm离心5min，则ETR沉入底部。

10.小心将上层液体转移至一新的15或10mL离心管中，加入0.5倍体积的EtOH（约2.5mL），轻柔混颠倒5-6次，室温静置2min。

11.将10中液体加入柱子中，室温4000rpm离心5min，弃液体，重新装好柱子，直至液体全部滤过。

omga质粒提取试剂盒_说明书_翻译

omgaE.Z.N.A.质粒试剂盒操作规程说明书（译版）1. 自新鲜划痕选择培养板中分离单菌落，接种含适当选择性抗生素的2-5ml的LB或YT培养基进行培养。

37℃强力摇动（~300rpm）孵育20-24h。

使用10-20ml培养管或容量至少4倍于培养容积的培养瓶。

强烈推荐使用endA阴性大肠杆菌菌株进行常规质粒分离。

此类菌株包括DH5α和JM109。

2. 将1.5-5.0ml细菌菌液室温13,000 g离心3min。

轻轻倒出或吸走培养基并丢弃。

3. 加入200μl 的溶液Buffer T 1/Rnase A重悬沉淀，涡旋或用移液器反复吹打。

充分重悬沉淀对于获得高质量的DNA非常重要。

4. 加入200μl溶液Buffer T 2，倒置、转动试管5-10次使之轻轻混匀，得到透明的裂解产物。

室温孵育5min。

不要用力混合，以免使染色体DNA断裂，减低质粒的纯度。

该步反应不要超过5min。

溶液II不用时要拧紧瓶盖，以免试剂被空气中的CO2酸化。

5. 加入200μl溶液Buffer T 3，立即倒置试管15-20次混匀，直至白色絮状沉淀物形成。

冰上孵育5min。

为避免形成局部沉淀，加入溶液III后应立即、充分混匀溶液。

6. 4℃13,000 g离心10分钟。

白色沉淀物形成，立即进行下一步操作。

7. 小心翼翼的吸取上清液，加入到新的1.5ml离心管（不是试剂盒提供的）中。

加入200ul 用异丙醇稀释的BAC 结合液（缓冲液），颠倒3-5次充分混匀。

BAC 结合液使用前必须用96%-100%的异丙醇稀释，详细的说明见瓶壁或第三页说明书8.加入样本DNA（HiBind DNA MicroElute）到提供的2ml收集管中。

9. 室温8000g离心30s，丢弃收集管，将离心柱放入新的收集管中。

10. 将收集柱放回收集管并加入750ul SPM buffer（用乙醇稀释），室温8000g离心30s，丢弃收集管，将离心柱放入新的收集管中。

Omega公司RNA提取试剂盒说明书

RNA-Solv Reagent®RNA Isolation SolventWARNING: This reagent is toxic if swallowed and in contact with skin. Causes burns. After contact with skin, wash immediately with copious amounts of mild detergent and water. If you feel sick, seek medical advice at once and Quote UN2821.Product No:R6830-00 (5 ml)R6830-01 (100 ml)R6830-02 (200 ml)Storage Conditions:RNA-Solv is stable for at least 24 months®when stored at 2°C-8°C and yields reproducible results.IntroductionRNA-Solv® Reagent is a reagent system for the isolation of total RNA from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step RNA isolation method developed by Chomczynski and Sacchi (1).The sample is homogenized and lysed in RNA-Solv® Reagent which maintains the integrity of the RNA, while disrupting and denaturing endogenous RNases and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. RNA remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X10), and large quantities of tissue ( up to1 g) and cells 6(<10 ), of human, animal, plant, or bacterial origin. The simplicity 8of the RNA-Solv® Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA prepared in this manner 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 amplification by thermal cycling, treatment of the isolated RNA with RNase-free DNase I is recommended when the two amplimers lie within a single exon.Supplied By User•Chloroform (no isoamyl alcohol added)•Isopropyl alcohol•80% Ethanol (in DEPC-treated water)•RNase-free water•Tabletop centrifuge capable of 12,000 x g at room temperatureGeneral Notes Regarding RNase ContaminationWhenever working with RNA :•Always wear disposable gloves and change gloves frequently.•Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases.•In the presence of RNA-Solv® Reagent, RNA is protected from RNase contamination. Downstream sample handling requires that nondisposable glassware or plasticware be RNase-free.•Use only DECP-treated buffers. Add DEPC to a final concentration of 0.1%, incubate at 37C for 2 hours, andoautoclave at 121C. Do not add DEPC to Tris buffers. Suchobuffers must be prepared by using DECP-water.PrecautionUse only disposable polypropylene tubes for small samples and glass Corex tubes for larger samples. All tubes must be able to withstand 12,000 x g . Polystyrene tubes may crack with chloroform Before StartingA.Small Samples :To isolate RNA from very small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) of samples in 0.8 mL of RNA-Solv®, and add 1 mg RNase-free glycogen or yeast tRNA as carrier. This will improve yields obtained with precipitation.B.Difficult Animal Samples: Specimens containing large amounts of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and sperm, will require the following modification. After lysis/homogenization in RNA-Solv® Reagent, centrifuge at 12,000 x g for 10 minutes at room temperature to remove insoluble debris. Often a precipitate forms at the bottom of the tube, but with fatty tissue, a lipid layer will also form above the aqueous phase. The supernatant will contain the RNA and must be carefully transferred to a fresh 1.5 ml microfuge tube before proceeding.C. Interruption the procedure:Following lysis in RNA-Solv®Reagent and before addition of chloroform, samples can be stored at -70C for up to 3 months. In addition, once the RNA is oprecipitated in isopropanol, the pellet may be stored at -20C or -o70C for up to 1 year.oRNA-Solv Protocol for Total RNA Isolation®CAUTION: When working with RNA-Solv® Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20C-25C).o o1. Homogenization and lysis of samples: follow either method belowa) Tissue SamplesHomogenize tissue samples in 1 mL of RNA-Solv® Reagent per 50-100 mg of tissue using an appropriate mechanical homogenizer. Alternatively one can pulverize tissue in liquid nitrogen with mortar and pestle and transfer the powder to a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (Eppendorf, Cat No. 0030 120.973; VWR, Cat No. KT 749520-0000). The sample volume should not exceed 10% of the volume of RNA-Solv® Reagent used.b) Cells Grown in SuspensionPellet cells by centrifugation. Lyse cells in RNA-Solv® Reagent by repetitive pipetting. Use 1 mL of the reagent per 5-10 x 10of6 animal, plant or yeast cells, or per 1 x 10 bacterial cells. Washing8cells before addition of RNA-Solv® Reagent should be avoided as this increases the possibility of mRNA degradation and RNase contamination. For plant, fungal, and yeast cells mechanical or enzymatic homogenization may be required. Also, for plant, fungal, and yeast cells, we recommend the use of the E.Z.N.A.® Plant (R6627),Fungal (R6640), and Yeast (R6670) RNA Kits from Omega Bio-tek.c) Cells Grown in MonolayerLyse cells directly in a culture dish by adding 1 mL of RNA-Solv®Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a blue pipette tip. The amount of RNA-Solv®Reagent added is based on the area of the culture dish (~1 mL per 10 cm ). An insufficient amount of RNA-Solv® Reagent may result2in contamination of the isolated RNA with DNA. Always use more RNA-Solv® Reagent if in the lysate is too viscous to aspirate with a pipette.2. Add 0.2 mL of chloroform per 1 mL of RNA-Solv® Reagent. Cap sample tubes securely and vortex vigorously for 15 seconds. Incubate on ice for 10 minutes. This step is critical - do not change it.3. Centrifuge the samples at no more than 12,000 x g for 15 minutes 4E C.The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA remains entirely in the aqueous phase.4. Precipitation of RNA.Transfer no more than 80% of the aqueous phase to a fresh tube, and discard the lower organic phase. Precipitate the RNA from the aqueous phase by adding 500ìl of isopropyl alcohol per 1 mL of RNA-Solv® Reagent used for the initial homogenization. Incubate samples at room temperature 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes also at room temperature.Carbohydrate-rich samples: Plant samples of high polysaccharide content or animal tissues rich in glycosaminoglycans (proteoglycans) require the following modified precipitation method for obtaining pure RNA. Prepare Buffer A ( 1.2 M sodium chloride, 800 mM sodium citrate). Following step 3, add to the aqueous phase 0.3 ml isopropanol followed by 0.3 ml Buffer A per 1 ml RNA Solv ® Reagent used in step 1. Vortex to mix and centrifuge at no more than 12,000 x g for 10 minutes at room temperature. This high salt precipitation will reduce co-purification of complex carbohydrates.5. Wash RNA pellet. Discard the supernatant and wash the RNA pellet once with 1 ml 80% ethanol. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at room temperature.6.Reconstitute RNA. Carefully aspirate and discard the ethanol and briefly AIR DRY the RNA pellet for 2-5 minutes at room temperature. Do not use centrifugal devices equipped with a vacuum source as over-drying will lead to difficulty in re-dissolving RNA in water. Dissolve RNA in RNase-free water - a 5 minute incubation at 60 °C may be required. RNA can also be reconstituted in 100% formamide (deionized) and stored at -70°C.RNA is now suitable for RNase protection, northern analysis and reverse transcriptase reactions. For isolation of poly(A)+ RNA an additional ethanol precipitation is required. Add 1/8 X volume of RNase-free 3M NaAc, pH 6.0 followed by 2.5 X volume absolute ethanol. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge at 12,000 x g for 10 min at room temperature and discard the supernatant. Wash the pellet as before and reconstitute in DECP-treated water.Determination of Yield and QualityUV spectrophotometric analysis of the purified RNA is required for obtaining yield. To do so, dilute the RNA in an appropriate volume of TE buffer, pH 8.0 (not water; RNA yields low Abs ratio values if dissolved in acidic buffers) and measure absorbance at 260 nm and at 280 nm. RNA Conc = 40 ìg/ml X Dilution factor X Abs 260 nmTypical Abs 260 nm/ 280 nm ratios of 1.7-1.9 are obtained with the protocol. Yields vary depending of type and amount of starting material, and on condition of storage prior to processing. For assessing the quality of RNA, we recommend you perform denaturing agarose gel electrophoresis to confirm the integrity of purified material. Invariably, the full spectrum of RNAs, including 4S and 5S species are purified with RNA-Solv® Reagent.Expected Yields per 1 mg tissue or 10 cells:6Liver and spleen, 5-10 ìgKidney, 2-5 ìgBrain, 1-2ìgEndothelial cells, 7-12 ìgFibroblasts, 6-8ìgTroubleshooting•Low RNA Yields: Incomplete lysis of samples in RNA Solv Reagent. RNA pellet not completelt dissolved in DEPC-water.pH of diluent used for spectrophotometric analysis is too low.•Degraded RNA: Tissues were not immediately processed or frozen. Inadequate storage of starting material prio to isolation. Inadequate storage of RNA (-5 to -20°C, instead of -60 to -70°C) Trypsin/EDTA was used in dislodging monolayer cells. Buffers or plasticwasre were not RNase-free.Formaldehyde used for denaturing agarose-gel electrophoresis had a pH below 3.0.•Low Abs260/Abs280 ratios: Sample was diluted in water rather than TE. Acidic pH lowers absorbance ratios. Use TE buffer as diluent for readings. Insufficient RNASolv Reagent was used for lysis of sample. Ice incubation in step 2 was not performed. The aqueous phase was contaminated with the phenolic phase.•DNA contamination of RNA: Too little RNASolv Reagent used for sample processing causing inadequate separation of DNA/nucleoprotein complexes from aqueous RNA. The aqueous phase was contaminated with the phenol phase.Technical Support:Omega Bio-tek, USA - call toll-free : 1 888 832 8896References:1. Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156 (1987).2. Chomczynski, P. Biotechniques 15, 532 (1993).For laboratory research use only.CAUTION: Not for diagnostic use. The safety and efficacy ofthis product indiagnostic or other clinical uses has not been established.May,1999 (C). All rights reserved by Omega Bio-tek, Inc.RNA-Solv is a registered mark of Omega Bio-tek, Inc.。

OMEGA Total RNA 提取试剂盒中文步骤

动物细胞总RNA提取步骤：注：所有的离心操作在室温中进行一般操作设备：1、完全乙醇（96~100%）2、无菌的移液枪头和1.5ml的离心管3、可选：14.3Mβ-mercaptoethanol (β-ME, 2-mercaptoethanol)4、14000Xg的微型离心机5、加入70%乙醇的DPEC水6、一次性手套二、样品破坏和均化设备1、omega均化柱2、注射器具3、研钵和碾槌4、转子定子均化器三、实验前准备：可选：每一毫升TRK Lysis Buffer加入20ul的2-mercaptoethanol)制成工作溶液。

混合液可在室温下保存一个星期。

1、hibind柱最大能收集100ug的RNA2、TRK Lysis Buffer能操作的最大细胞数为1X10^7Source Number of Cells RNA Yield (μg)IC21 1 x 106 12Hela 1 x 106 15293HEK 1 x 106 10HIN3T3 1 x 106 15四、收集细胞1、收集悬浮细胞:细胞数数，500g离心5分钟沉淀细胞，吸掉上清，继续下一步2，收集单层细胞（1）直接溶解细胞：细胞数数，吸掉全部的完全培养基，继续下一步（2）胰蛋白酶消化收集细胞细胞数数，吸掉培养基，PBS洗几遍。

用加了0.1~0.25%的胰蛋白酶的PBS消化细胞，当细胞从培养皿分离，加入培养基终止消化。

500g离心5分钟，吸掉上清，继续下一步。

五、用TPK Lysis Buffter 破坏细胞1对于细胞团，摇晃收集管使细胞团松散，按下表加入适量的TRK Lysis Buffer,振荡混匀。

2、对于在培养板上的单层细胞，按下表加入适量的TRK Lysis Buffer,，用rubber policeman 收集细胞并转移至1.5ml的离心管中，振荡或吹打混合。

六、按以下其中一种方法匀浆化和破坏组织1、Rotor-Stator 匀浆机：用匀浆机使样品完全均匀。

细菌RNA提取方法-OMEGA试剂盒法

1.取3mL菌体，6000g离心5min，4°C，弃上清，加入500μL 1×TE Buffer，用枪头吹吸
均匀，6000g离心5min，4°C，弃上清。

2.加入100μL 溶有溶菌酶的TE Buffer（20mg/mL），重悬菌体，涡旋震荡30s。

3.30°C水浴10min，每隔2min涡旋震荡20s。

4.加入350μL BRK buffer（第一次使用之前加入β-巯基乙醇，使其浓度为20mg/mL），加
入25-40mg玻璃粉，涡旋震荡5min，13000g离心5-10min。

5.转移400μL上清至新的EP管中，加入400μL 70%冰乙醇，用枪头轻轻混匀。

6.将液体（包括可能生成的沉淀）转移至柱子中，10000g离心1min，弃废液。

7.加入300μL Wash Buffer I，10000g离心1min，弃废液。

8.加入500μL Wash Buffer I，10000g离心1min，弃废液。

9.加入500μL Wash Buffer II，10000g离心1min，弃废液。

10.重复步骤9。

空转2min。

11.将柱子转移至新的EP管中，加入30-50μL DEPC水，10000g离心1min。

如需要可重复一
次。

DEPC水可提前70°C预热。

RNA提取-OMEGA

OMEGA E.Z.N.A. TM RNA 提取试剂盒操作步骤1.弃培养基，加入1ml RNA-Slov regent 裂解细胞，将裂解液转移到一干净的1.5ml EP管中，室温孵育2-3min。

2.每1ml RNA-Slov regent中加入200µl氯仿，涡旋混匀15s，冰上孵育10min。

3.12,000×g，4 ℃，离心15min。

4.吸取上层水相并加入1/3体积的无水乙醇，涡旋混匀15s。

5.将HiBind RNA Spin柱子套在2ml收集管中，该柱子一次性最多可以容纳700µl的溶液。

将上述溶液（包括沉淀）转移至柱子上，室温下，10,000×g离心1min，以完全滤过溶液。

如果还有溶液残留，请延长时间至溶液完全滤过柱子，去弃滤过液。

继续将剩余的裂解液转移至柱子，离心弃去滤出液。

Tip：如果在离心后，柱子发生堵塞，可延长离心时间或提高离心速率至裂解液完全流出柱子。

柱子发生堵塞的原因有以下几种原因：1.样品起始量太多；2.裂解不够充分；建议：1.减小样品量，提高匀浆效率；2.裂解完后，用匀浆柱过滤裂解液。

6.将柱子放入一个新的2ml收集管中，加入300µl Wash Buffer I至柱子上，室温下，10,000×g离心1min，去除滤过液；7.将柱子放置于2ml收集管中，加入400µl RNA Wash Buffer I，室温下，10,000×g离心1min，去除滤过液；8.将柱子套回离心管，加入500µl RNA Wash Buffer II(已用无水乙醇稀释)，室温下，10,000×g离心1min，弃去滤过液；9.将柱子套回离心管，加入500µl RNA Wash Buffer II，室温下，10,000×g离心1min，弃去滤过液。

10.将柱子套回空的收集管，室温下，14,000×g离心空柱2 min，以甩干柱子的基质。

OMG植物RNA提取试剂盒中文使用说明

OMG植物RNA提取试剂盒中文使用说明植物RNA提取试剂盒（Plant RNA Extraction Kit）是一种高效的试剂盒，用于从植物组织样品中提取RNA。

本文将详细介绍该试剂盒的使用说明，以帮助用户正确操作。

步骤一：样品的预处理1.1收集所需的植物组织样品，并将其快速冷冻在液氮中。

1.2在试验台上，用磨砂盘将样品研磨成粉末状。

使用过程中，要避免样品的过度加热以及溶液的损失。

1.3 从研磨后的样品中取出约100 mg的样品，放入2 mL离心管中。

步骤二：细胞破碎和裂解2.1向离心管中加入1mL细胞破碎液，然后快速颠倒数次，使样品均匀悬浮。

2.2将悬浮液在65摄氏度孵育30分钟，每隔5分钟振荡一次。

2.3孵育结束后，将管子在高速离心机中离心5分钟，以去除细胞残渣。

步骤三：RNA的沉淀3.1将上一步得到的上清液转移至新的2mL离心管中。

3.2加入1.5mL乙醇，轻轻颠倒数次，混合均匀。

3.3将混合液转移至RNA沉淀柱中，并在2mL收集管中储存过滤液。

3.4在6,000×g的离心机中离心柱子30秒，将上清液排除。

3.5在柱子上方放置一个新的2mL离心管，并将柱子放在上面。

3.6加入700µLRNA洗涤缓冲液，然后在6,000×g的离心机中离心柱子1分钟，将上清液排除。

3.7在同一个离心管中，再次加入700µLRNA洗涤缓冲液，然后在6,000×g的离心机中离心柱子1分钟，将上清液排除。

步骤四：RNA的洗涤和干燥4.1将柱子转移到新的2mL离心管中。

4.2加入600µL高盐洗涤缓冲液，然后在6,000×g的离心机中离心柱子1分钟，将上清液排除。

4.3在同一个离心管中，再次加入600µL高盐洗涤缓冲液，然后在6,000×g的离心机中离心柱子1分钟，将上清液排除。

4.4将柱子转移到新的1.5mL离心管中。

4.5 加入30 µL RNase-free水溶解RNA，然后在常温孵育5分钟。

promega质粒提取试剂盒操作说明（中文）

promega质粒提取试剂盒操作说明（中⽂）注意:所有纯化和洗脱步骤均在室温下进⾏。

1. 将转化⼤肠杆菌细胞在最佳培养条件下培养50-250ml过夜(16 - 21h)。

注意:本⽅案优化为过量培养50-250ml，OD600 = 2-4。

2. 将细菌收集起来，5000 g离⼼10分钟，弃上清。

⽤纸⼱除去多余的介质。

表1 解液所需的溶液体积。

3. 在细胞重悬液中重悬细菌。

4. 加⼊细胞裂解液和轻轻混合翻转管3-5次或混合裂解液轻轻滚动。

室温下孵育3分钟。

注:如果细胞裂解液过冷，可能会发⽣SDS沉淀，导致细胞裂解不良。

如果有沉淀形成，将细胞裂解液加热到37°C，并轻轻摇晃。

5. 在溶解细胞中加⼊中和液，盖住并轻轻翻转5-10次。

6. 15000g室温离⼼15分钟。

这种离⼼法将使颗粒化⼤量的细菌碎⽚。

使⽤PureYield™清除柱清除剩余碎⽚。

要区分PureYield™Clearing和PureYield™Binding列，请注意过滤管是蓝⾊的，⽽结合柱是⽩⾊的。

7. 通过在PureYield™顶部嵌套⼀个PureYield™(蓝⾊)组装。

如图1所⽰，将组装好的柱堆放在真空泵上。

图1所⽰。

蓝⾊清除列插⼊⽩⾊绑定列。

8. 将澄清的裂解液倒⼊PureYield™澄清柱中。

不要让颗粒状的碎⽚落⼊管内。

9. 启动真空泵。

裂解液将通过PureYield™澄清柱中的澄清膜，并且 DNA将在PureYield™结合柱中结合到结合膜上。

继续抽吸所有的液体都通过了两个柱。

注意:所有纯化和洗脱步骤均在室温下进⾏。

10. 在进⾏前，从过滤装置中缓慢释放真空。

移除PureYield™清除柱，将PureYield™结合⾊谱柱留在泵上。

注意:如果真空释放得太快，膜可能会从柱上分离。

如结合膜脱落，⽤带⼿套的⼿指或1.0ml⽆菌溶液的⼤端轻轻轻拍移液器尖端，或打开真空，让压⼒重新安置膜。

11. 向柱中加⼊5.0ml的内毒素清除液，让真空泵将溶液穿过。

OMEGArna提取试剂盒中文说明

OMEGArna提取试剂盒中文说明E.Z.N.A. T otal RNA Kit I (R6834-01 50 preps)步骤A ．真核细胞和组织自己需要的材料：β－巯基乙醇、70％乙醇（DEPC 水配置）、除RNase 的枪头和EP 管。

材料的最佳量想得到最佳的产量和好的Hibind RNA 柱的纯化效果，选用正确的细胞和组织量是最重要的。

Hibind RNA 柱的最大处理量是可变的，根据细胞和组织的类型。

最大的结合能力是100ug 的RNA 。

TRK 裂解缓冲液的最大裂解能力是1*107个细胞或30mg 的组织。

细胞的步骤1．用500ul 的TRK 裂解缓冲液裂解细胞（≤1*107），使细胞团松散，轻轻弹EP 管或者用枪头吹旋，再用漩涡振荡混匀。

a) 使用前每1ml TRK 裂解缓冲液加入20ul 的β－巯基乙醇。

b) 如果是单层的组织培养细胞（成纤维细胞，内皮细胞等），可以直接在细胞培养器里裂解细胞。

完全吸去培养基后直接加TRK 裂解缓冲液至细胞中。

加700ul 的TRK 到T35细颈瓶或10cm 的培养皿中，更小的培养器加500ul 的TRK 。

将液体布满整个器皿表面，确保细胞裂解完全。

将裂解产物转移至1.5mlEP 管中。

c) 如果细胞是悬液培养，收集细胞（≤1,500rpm or 400*g ）*5min ，弃上清，加入TRK裂解液，漩涡振荡混匀。

2．匀浆化裂解产物“裂解和匀浆化样品”－第四页有详细的说明，如果细胞≤1*105，漩涡振荡1min 。

不完全使样品匀浆化会显著的减少RNA 的产量还容易造成柱子堵塞。

a) 用匀浆器30 s ，使样品匀浆化。

b) 用钝的针头的注射器（0.9mm 直径），至少吹打5次是样品匀浆化。

注射器要除RNase 。

3．将匀浆后的裂解产物转移至Homogenization Spin Column 中，离心，≥1,4000*g, 3min,室温。

将离心收集的流出液转移到新的EP 管中。

OMEGA Total RNA 提取试剂盒R6834说明书

E.Z.N.A.® Total RNA Kit ITable of Contents Introduction (2)Illustrated Protocol (3)Kit Contents/Storage and Stability (4)Preparing Reagents (5)Recommended Settings (6)Quantification of RNA (7)Disruption and Homogenization (8)Animal Cell Protocol (10)Animal Tissue Protocol (13)Spin/Vacuum Manifold Protocol (16)DNase Digestion Protocol (18)Troubleshooting Guide (20)Ordering (22)Manual Revision: October 2009Innovations in nucleic acid isolation1IntroductionThe E.Z.N.A.® RNA family of products is an innovative system that radically simplifies the extraction and purification of RNA from a variety of sources. The key to this systemis that it uses the reversible binding properties of the HiBind Matrix in combinationwith the speed of mini-column spin technology thereby permitting single or multiple simultaneous processing of samples. There is no need for phenol/chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation, or precipitation with isopropanol or LiCl are eliminated. RNA purified using the E.Z.N.A.® RNA Purification System is ready for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.The E.Z.N.A.® Total RNA Kit I can purify up to 100 μg of total RNA from cultured eukaryotic cells or tissue. Normally, 1 x 106 - 1 x 107 eukaryotic cells, or 5-30 mg of tissue, can be processed in a single experiment. Fresh, frozen or RNALater® stabilized tissues can be used. Lysis of cells or tissue occurs under denaturing conditions which inactivate RNases. After the homogenization process, samples are applied to the HiBind RNA spin column to which total RNA binds. Cellular debris and other contaminants are effectively washed away after a few quick wash steps. High quality RNA is finally eluted in DEPC treated water. Total RNA greater than 200 nt is isolated using this kit.For isolating total RNA below 200 nt use the miRNA isolation Kit (R7034). For isolating total RNA from gram positive bacteria, the recommended kit is the Bacterial RNAKit(R6950).While this kit may be used for the isolation of RNA from whole blood, we recommend that you use the E.Z.N.A.® Blood RNA Kit (Product # R6814) as it is specifically designed for effective hemolysis and hemoglobin removal, thereby giving higher RNA yields. Binding CapacityEach HiBind RNA Mini column can bind approximately 100μg of RNA. Using greater than 30 mg of tissue or 1 x 107 cells is not recommended.23Kit ContentsStorage and StabilityAll E.Z.N.A.® Total RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature. During shipment, crystals or precipitation may form in the TRK Lysis Buffer. Dissolve by warming buffer to 37°C.4Preparing Reagents• Dilute RNA Wash Buffer II with absolute ethanol (96-100%) as follows:• Store diluted RNA Wash Buffer ll at room temperature.• Please remember to always wear gloves whenever working with RNA. This will minimize RNase contamination. Use only clean RNase-free disposable plastic pipette tips when us-ing the supplied reagents.• Under cool ambient conditions, crystals may form in TRK Lysis Buffer. This is normal; warm at 37°C to dissolve.• Optional: As a preparation step add 20µl of 2-mercaptoethanol (β-mercaptoethanol) per 1 ml of TRK Lysis Buffer in order to achieve a working solution. This mixture can be stored for 1 week at room temperature.5Guideline for Vacuum ManifoldThe following is required for use with the Vacuum Protocol:A) Vacuum Manifold (We recommend Omega Bio-tek’s VAC-08)Other Compatible Vacuum Manifolds: Qiagen QIAvac24, Sigma AldrichVM20, Promega Vacman®, or manifold with standard Luer connectorB) Vacuum FlaskC) Vacuum TubingD) Vacuum Source (review tables below for pressure settings)B) Vacuum Flask D) Vacuum SourceIllustrated Vacuum Setup:67Quantification of RNAStorage of RNAPurified RNA can be stored at -70°C (RNase-free water). Under such conditions, RNA prepared with the E.Z.N.A.® Total RNA Kit l is stable for more than a year.Quantification of RNATo determine the concentration and purity of RNA, one should measure theabsorbance at 260 nm and 280 nm in a spectrophotometer. 1 O.D. unit measured at 260 nm corresponds to 40μg of RNA per ml. DEPC treated water is slightly acidic and can dramatically lower absorbance values. We suggest that you dilute thesample in a buffered solution (TE) for spectrophotometric analysis. The A 260/A 280 ratio of pure nucleic acids is 2.0, while for pure protein is approximately 0.6. Therefore, a ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid. [Phenol has a maximum absorbance at 275 nm and can interfere with absorbance readings of DNA or RNA.However, the E.Z.N.A.® Total RNA Kit eliminates the use of phenol and avoids this problem.]RNA QualityIt is highly recommended that RNA Quality be determined prior to all analysis. The quality of RNA can be best assessed by denaturing agarose gel electrophoresis and ethidium bromide staining. Two sharp bands should appear on the gel. These are the 28S and the 18S (23S and 16S for bacteria) ribosomal RNA bands. If these band appears as a smear towards lower molecular weight sized RNAs, the it is likely that RNA has undergone major degradation during preparation, handling, or storage.Although RNA molecules less than 200 bases in length do not efficiently bind to the HiBind matrix, a third RNA band, the tRNA band, may be visible when a large number of cells are used.Expected YieldsFor animal cell yields, see page 9.For animal tissue yields, see page 14.Disruption and Homogenization of SamplesEfficient sample disruption and homogenization is essential for successful Total RNA isolation. Complete cell wall and plasma membrane disruption is very important for the release of all of the RNA contained in the sample. The purpose of homogenization is to reduce the viscosity of the cell lysates produced by cell disruption. Homogenization shears genomic DNA and other high molecular weight cell components thereby creating a homogenous lysate. Incomplete homogenization will cause RNA binding to clog thus preventing efficient RNA binding result in low or no yield.Mortar and Pestle: Sample DisruptionSample disruption using a mortar and pestle followed the chosen of homogenization method:Wear gloves, and take great care when working with liquid nitrogen.1. Excise tissue and promptly freeze in a small volume of liquid nitrogen.2. Grind tissue with a ceramic mortar and pestle under approximately 10 ml of liquidnitrogen. Pour the suspension into a pre-cooled 15ml polypropylene tube. The tube must be pre-cooled in liquid nitrogen or the suspension will boil vigorously possibly causing tissue loss.3. Allow the liquid nitrogen to completely evaporate and add TRK lysis buffer. Homogenization:A) Homogenizer Spin column (Product # HCR 003)Load the lysate into a homogenizer spin column pre-inserted into a 2ml collection tube. Spin for two minutes at maximum speed in a micro centrifuge in order tocollect homogenized lysate.B) Syringe and NeedleShear High molecular-weight DNA by passing the lysate through a narrowneedle (19-21 gauge) 5-10 times.Rotor-Stator Homogenizer: Sample Disruption and HomogenizationUsing a rotor-stator homogenizer for sample disruption and homogenization can simultaneously disrupt and homogenize most samples. The process usually takes less than a minute depending on sample type. Many Rotor-stator homogenizers operate with differently sized probes or generators that allow sample processing in 50ml tubes. Bead Milling: Sample Disruption and HomogenizationBy using bead milling, cells and tissue can be disrupted and homogenized byrapid agitation in the presence of glass beads and a lysis buffer. The optimal amount of glass beads to use for RNA isolation are 0.5mm for yeast/unicellular cells and 4-8mm for animal tissue samples.8Total RNA Kit I - Animal Cell ProtocolE.Z.N.A.® Total RNA Kit I Animal Cell ProtocolAll centrifugation steps used are preformed at room temperature.General Protocol Equipment:• Absolute(~96-100%) Ethanol• Sterile RNase-free pipet tips and 1.5ml centrifuge tubes• Optional: 14.3M β-mercaptoethanol (β-ME, 2-mercaptoethanol)• Microcentrifuge capable of at least 14,000 x g• 70% ethanol in Sterile DEPC-treated Water.• Disposable glovesSample Disruption and Homogenization Equipment:• Omega Homogenizer Columns (HCR003)• Needle and Syringe• Mortar and pestle• Glass Beads• Rotor-stator HomogenizerThings to do before starting:• Optional: As a preparation ste p add 20μl of 2-mercaptoethanol (β-mercaptoethanol) per 1 ml of TRK Lysis Buffer in order to achieve a working solution. This mixture can be stored for 1 week at room temperature.1. Determine the proper amount of starting material: It is critical to use the correctnumber of starting cells in order to obtain optimal yield and purity with the HiBind RNA column. The maximum number of cells that can be processed on a HiBindRNA column is dependent on the specific RNA contents and type of cell line. Themaximum binding capacity of the HiBind RNA column is 100µg. The maximumnumber of cells that TRK Lysis Buffer can use in the Total RNA Protocol is 1 x 107. Use the following table as a guideline to select the correct starting material.Expected Yield9102.Harvest cells by choosing one of the following methods (A or B).(do not use more than 1 x 107 cells)A) For cells grown in suspension: determine the number of cells. Pellet theappropriate number of cells by centrifuging at 500 x g for 5 minutes. Aspirate the supernatant and continue with step 3 of this protocol. OrB) For cells grown in a monolayer: These cells can either be lysed directly in the cell culture dish or trypsinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should always be trypsinized.i. For direct cell lysis:Determine cell number, and aspirate the cell-culture medium completely. Immediately proceed to step 3.NOTE: Not removing cell-culture medium completely will inhibit lysis anddilute the lysate. This will affect the conditions for binding of RNA to the HiBind RNA column, and may reduce RNA yield.ii. To trypsinize and collect cells:Determine cell number. Aspirate the medium and wash cells with PBS. Aspirate the PBS, add 0.1-.25% trypsin into PBS. Add medium (containing serum to inactivate the tryspin), after the cells detach from the dish or flask. Transfer cells to an RNase-free glass or polypropylene centrifuge tube (not supplied), and centrifuge at 500 x g for 5 minutes. Aspirate the supernatant completely, and proceed to step 3.Note : Not removing cell-culture medium completely will inhibit lysis and dilute the lysate. This will affect the conditions for binding of RNA to the HiBind RNA column, and may reduce RNA yield.3.Disrupt cells (do not use more than 1 x 107 cells) with TRK Lysis Buffer.For pelleted cells, loosen the cell pellet thoroughly by flicking the tube, and adding the appropriate amount of TRK Lysis Buffer based on the table below. Mix throughlyby vortexing or pipetting.Total RNA Kit I - Animal Cell ProtocolFor direct lysis of cells grown in a monolayer, add the appropriate amount of TRK Lysis Buffer directly to the cell culture dish based on the table below. Collect the cell lysate with a rubber policemen and transfer the cell lysate into a 1.5 ml centrifuge tube. Mix throughly by vortexing or pipetting. Make sure there is no visible cell clumps in the sample.4. Homogenize and disrupt the tissue accordingly to one of the following steps:1. Rotor-Stator Homogenizer: Homogenize cells with a rotor-stator homogenizer oruntil the sample is uniformly homogenized. See Page 8 for details.2. Syringe and Needle: S hear high molecular-weight DNA by passing the lysate through a narrow needle (19-21 gauge) 5-10 times.3. Omega Homogenizer Column (HCR003): Load the lysate into a homogenizer spin column pre-inserted into a 2ml collection tube. Spin for two minutes at maximum speed in a micro centrifuge in order to collect homogenized lysate. Note : Incomplete homogenization of the sample will cause clogging colum thus resulting lower yields or no yield. It is recommended to homogenize the tissue sample with rotor-stator homogenizers since it normally produces better yields. 5.Add an equal volume (350µl or 700µl) of 70% ethanol to the lysate and mixthoroughly by pipetting up and down 3-5 times. Do not centrifuge. If any sample has lost its volume during homogenization, adjust the volume of ethanol accordingly. Note: During RNA purification from certain cell lines, a precipitate may form after the addition of ethanol. This does not affect the procedure. 6.Apply the sample ( including any precipitate that may have formed) to a HiBind RNA column inserted into a 2ml collection tube (supplied). Centrifuge at 10,000 x g for 60 seconds at room temperature. Discard flow-through and reuse the collection tube in the next step.Note: The maximum capacity of the HiBind RNA spin column is 700µl. Largervolumes can be loaded on to the column successively in the same HiBind RNA spin column. Discard flow-through after each centrifugation.Optional: This is the starting point if performing the optional on-column DNase Idigestion . Follow protocol as outlined on page 18, after completing this step.Total RNA Kit I - Animal Cell ProtocolTotal RNA Kit I - Animal Cell Protocol7. Add 500µl of RNA Wash Buffer I by pipetting directly onto the HiBind RNA column.Centrifuge at 10,000 x g for 60 seconds. Discard the flow-through and reuse thecollection tube in the next step.8. Add 500µl of RNA Wash Buffer II by pipetting directly onto the HiBind RNA column.Centrifuge at 10,000 x g for 60 seconds at room temperature. Discard flow-through and reuse the collection tube in the next step.Important: RNA Wash Buffer II must be diluted with absolute ethanol before use.Refer to label for instructions. If refrigerated, RNA Wash Buffer II must be brought to room temperature before use.9. Add 500µl of RNA Wash Buffer II by pipetting directly onto the HiBind RNA column.Centrifuge at 10,000 x g for 60 seconds at room temperature and discard flow-through and place the HiBind RNA column in a new 2 mL collection tube(supplied).10. Centrifuge the HiBind RNA column for 2 minutes at maximum speed to completelydry the HiBind matrix.11. Elution of RNA: Transfer the column into a clean 1.5 ml centrifuge tube (notsupplied), and elute the RNA by adding 40-70µl of DEPC-treated water (supplied).Make sure that you add the water directly onto the center of the column matrix.Centrifuge for 1 minute at maximum speed. A second elution may be necessary if the expected yield of RNA is > 30 µg.Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration of RNA will be lower since more than 80% of RNA has been recovered in the first elution.Total RNA Kit I - Animal Tissue ProtocolE.Z.N.A.® Total RNA Kit I Animal Tissue ProtocolAll centrifugation steps used are preformed at room temperature.General Protocol Equipment:• Absolute(~96-100%) Ethanol• Sterile RNase-free pipet tips and 1.5ml centrifuge tubes• Optional: 14.3M β-mercaptoethanol (β-ME, 2-mercaptoethanol)• Microcentrifuge capable of at least 14,000 x g• 70% ethanol in Sterile DEPC-treated Water.• Disposable latex glovesSample Disruption and Homogenization Equipment:• Liquid Nitrogen• Omega Homogenizer Columns (HCR 003)• Needle and Syringe• Mortar and pestle• Glass Beads• Rotor-stator HomogenizerBefore starting:• Optional: As a preparation step add 20µl of 2-mercaptoethanolβ-mercaptoethanol)per 1 ml of TRK Lysis Buffer in order to achieve a workingsolution. This mixture can be stored for 1 week at room temperature.1. Determine the proper amount of starting material: It is critical to use the correcttissue amount in order to obtain optimal yield and purity with the HiBind RNAcolumn. The maximum amount of tissue that can be processed on a HiBind RNAcolumn is dependent on the specific RNA contents and type of tissue. The maximum binding capacity of the HiBind RNA column is 100μg. The maximum amount of tissue that TRK Lysis Buffer can lyse in the this protocol is 30mg. Use the following table as a guideline to select the correct starting material. If no information regarding starting material is available, use 10 mg as a starting amount. Given the yield and quality of RNA obtained from 10 mg, adjust the starting amount in the next purification.Average Yield of Total Cellular RNA From Mouse TissueAmount of TRK Lysis Buffer to be added in step 2 based on the table below. ForSamples stored in RNALater® use 700 μL of TRK Lysis Buffer.2.Homogenize and disrupt the tissue accordingly to one of the following steps:A. Rotor-Stator Homogenizer: Add the appropriate amount of TRK lysis buffer to the sample. Homogenize tissue with a rotor-stator homogenizer or until the sample is uniformly homogenized. (see Page 8 for details)B. Disrupt with Mortar and Pestle then homogenize with one of the following methods : Place the tissue in liquid nitrogen and grind using a mortar and pestle. Transfer the liquid nitrogen and tissue to a 1.5 mL centrifuge tube (not supplied). Let the liquid nitrogen evaporate without allowing the tissue sample to thaw. Add the appropriate amount of TRK lysis buffer to the grounded ample. Then homogenize with one of the following methods:l. Transfer the lysate to an Omega Homogenizer column (HCR003) pre-inserted intoa 2 mL collection tube. Centrifuge at >13,000 x g for 2 minutes. ll. Pass the lysate through a narrow needle (19-21 gauge) 5-10 times.Note : Incomplete homogenization of the sample will cause clogging colum thusresulting lower yields or no yield. It is recommended to homogenize the tissue samplewith rotor-stator homogenizers since it normally produces better yield.Total RNA Kit I - Animal Tissue ProtocolTotal RNA Kit I - Animal Tissue Protocol3. Centrifuge at full speed (≥ 13,000 x g) for 5 minutes. Carefully transfer the clearedsupernatant by pipetting to a clean 1.5 ml centrifuge tube (not supplied). Use only this supernatant (lysate) in subsequent steps.Note: In some preparations, a fatty upper layer will form after centrifugation.Transferring any of the fatty upper layer may reduce RNA yields or clog the column.4. Add an equal volume (350µl or 700µl) of 70% ethanol to the lysate and mixthoroughly by pipetting up and down 3-5 times. Do not centrifuge. If any sample has lost its volume during homogenization, adjust the volume of ethanol accordingly.Note: A precipitate may form after the addition of ethanol in certain preparations.This does not affect the procedure.5. Apply the sample ( including any precipitate that may have formed) to a HiBind RNAspin column placed into a 2ml collection tube (supplied). Centrifuge at 10,000 x g for60 seconds at room temperature. Discard flow-through and reuse the collection tubein the next step.Note: The maximum capacity of the HiBind RNA spin column is 700 µl. Largervolumes can be loaded on to the column successively in the same HiBind RNA spin column. Discard flow-through after each centrifugation.Optional: This is the starting point if performing the optional on-column DNase I digestion. Follow protocol as outlined on page 18, Immediately after completion of step6. Add 500 µl of RNA Wash Buffer I by pipetting directly onto the HiBind RNA Column.Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through and reuse thecollection tube in the next step.7. Add 500 µl of RNA Wash Buffer II by pipetting directly onto the HiBind RNA Column.Centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flow-through and reuse the collection tube in the next step.Important: RNA Wash Buffer II must be diluted with absolute ethanol before use.Refer to label for instructions. If refrigerated, RNA Wash Buffer II must be brought to room temperature before use.8. Add 500 µl of RNA Wash Buffer II by pipetting directly onto the HiBind RNA Column.Centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flow-through and place the HiBind RNA column in a new 2 mL Collection tube(supplied)..9. With the empty 2 ml collection tube, centrifuge the HiBind RNA column for 2 minutesat maximum speed to completely dry the HiBind matrix.10. Elution of RNA: Transfer the column into a clean 1.5 ml centrifuge tube (notsupplied), and elute the RNA with 40-70µl of DEPC-treated water (supplied). Make sure that you add the water directly onto the center of the column matrix. Centrifuge for 2 minutes at maximum speed (≥ 13,000 xg). A second elution may be necessary if the expected yield of RNA is > 30 µg.E.Z.N.A.® Total RNA Kit Vacuum/Spin ProtocolCarry out lysis, homogenization steps as indicated in previous protocols. Instead of continuing with centrifugation, follow the steps below. Do not use more than 106 cellsor 10 mg of tissue for the vacuum protocol.Note: Please read through previous section of this manual before proceeding withthis protocol.User Supplied Equipment:• Vacuum manifold• Vacuum sourceThings to do before starting:• Assemble vaccum manifold (see page 6)1. Prepare the vacuum manifold according to manufacturer’s instruction and connectthe HiBind RNA Column to the manifold.2. Load the homogenized sample onto the HiBInd RNA column.Note: Steps 1-4 from the Total RNA Animal Cell protocol should be completed orsteps 1-4 from the Total RNA Animal Tissue Protocol should be completed beforeloading the sample to the HiBind RNA column.3. Switch on the vacuum source to draw the sample through the column.4. Add 500 µl of RNA Wash Buffer I, draw the buffer through the column by turningon the vacuum source. Turn off the vacuum source when the buffer has beencompletely drawn through the column.5. Add 500 µl of RNA Wash Buffer II, draw the buffer through the column by turning onthe vacuum source. Turn off the vacuum source when the buffer has been completely drawn through the column.Important: RNA Wash Buffer II must be diluted with absolute ethanol before use.Refer to label for instructions. If refrigerated, RNA Wash Buffer II must be brought to room temperature before use.6. Add another 500 µl of RNA Wash Buffer lI, draw the buffer through the column byturning on the vacuum source. Turn off the vacuum source when the buffer has been completely drawn through the column.7. Assemble the column into a 2 ml collection tube(supplied) and transfer the columnto a micro centrifuge. Centrifuge at full speed for 2 minutes to dry the column matrix.8. Place the column in a clean 1.5 ml microcentrifuge tube (not supplied), and add40-70µl of DEPC-treated water (supplied). Make sure that you add the water directly onto the center of the column matrix. Centrifuge for 2 minutes at 10,000 x g. Asecond elution may be necessary if the expected yield of RNA is > 30 µg.E.Z.N.A.® Total RNA Kit I DNase Digestion ProtocolFor most downstream applications it is not necessary to do DNase digestion due to HiBind RNA resin and spin column technology removing nearly all DNA without the need for DNase Treatment. However, certain sensitive RNA applications might require further removal of DNA. In such case, we recommend that you please follow the outlined steps below using product E1091.Note: After completing steps 1-5 of either of the centrifugation protocols or steps 1-2 of the vacuum protocol (making sure that all of the sample has completely passed through the HiBind RNA column), proceed with the following steps.All centrifugation steps used are preformed at room temperature.User Supplied Material:• RNase Free DNase Set (E1091)1. For each HiBind RNA column, prepare the DNase I stock solution as follows: ArrayNote:• DNase I is very sensitive to physical denaturation, therefore do not vortex this DNase I mixture. Please mix by GENTLY inverting the tube. Remember to freshlyprepare DNase I stock solution right before RNA isolation.• E.Z.N.A.® DNase I Digestion Buffer is supplied with Omega Bio-Tek, Inc.’s RNase-Free DNase Set (product no. E1091). Standard DNase Buffers are not compatiblewith on-membrane DNase digestion. The use of other buffers may affect thebinding of RNA to the HiBind matrix, reducing RNA yields, and purity.2. Add 250µl of RNA Wash Buffer I by pipetting directly onto a new HiBind RNAcolumn inserted in a 2 ml collection tube. Centrifuge at 10,000 x g for 60 seconds anddiscard the flow-through and collection tube.3. Place the RNA column into a new 2 ml collection tube. Pipet 75µl of the DNase I stocksolution directly onto the surface of the HiBind RNA resin in each column. Make sure to pipet the stock solution directly onto the center of membrane. DNase I Digestion will not go through completion if some of the stock solution remains stuck to the wall or the o-ring of the HiBind RNA column.4. Incubate at room temperature (25-30°C) for 15 minutes.5. Add 500 μl of RNA Wash Buffer I. Place the column on a bench top for 2 minutes.Centrifuge at 10,000 x g for 60 seconds and discard flow-through and reuse thecollection tube.6. Add 500μl of RNA Wash Buffer II. Centrifuge at 10,000 x g for 60 seconds and discardflow-through and reuse the collection tubeImportant: RNA Wash Buffer II must be diluted with absolute ethanol before use.Refer to label for instructions. If refrigerated, RNA Wash Buffer II must be brought to room temperature before use.7. Add another 500μl of RNA Wash Buffer II. Centrifuge at 10,000 x g for 60 seconds anddiscard flow-through and reuse the collection tube.8. With the empty collection tube centrifuge the HiBind matrix for 2 minutes atmaximum speed to completely dry the HiBind matrix.9. Place the column in a clean 1.5 ml microcentrifuge tube (not supplied), and add40-70µl of DEPC-treated water (supplied). Make sure to add water directly onto the center of the column matrix. Let it sit for 1 minute, and then centrifuge for 2 minutes at maximum speed to elute the RNA. A second elution may be necessary if theexpected yield of RNA > 30μg.Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lower since more than 80% of RNA has been recovered in the first elution.Troubleshooting GuidePlease use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free,at (800-832-8896).Possible Problems and SuggestionsTroubleshooting GuideOrdering InformationThe following components are available for purchase separately.(Call Toll Free at 1-800-832-8896)PCR is a patented process of Hoffman-La Roche. Use of PCR process requires a license.Qiagen ®, QIAvac ® and Vacman ® are all trademarks of their respected companies. RNALater is a trademark of Ambion, IncHibind, E.Z.N.A and MicroElute are registered trademarks of Omega Bio-tek, Inc.。

OMEGA总RNA提取试剂盒说明书_BBH

E.Z.N.A. Total RNA Kit I (R6834-01 50 preps) 步骤A．真核细胞和组织自备材料：β－巯基乙醇、70％乙醇（DEPC水配置）、除RNase的枪头和EP管、一次性乳胶手套。

细胞的步骤1．在离心管中用TRK裂解缓冲液裂解细胞，使细胞团松散，轻弹EP管或者用枪头吹旋，再用漩涡振荡混匀。

使用前每1ml TRK裂解缓冲液加入20ul的β－巯基乙醇。

如果是单层的组织培养细胞（成纤维细胞，内皮细胞等），可以直接在细胞培养器里裂解细胞。

吸净培养基后直接加TRK裂解缓冲液至细胞中。

加600ul的TRK到T35细颈瓶或10cm的培养皿中，更小的培养器则加350ul的TRK。

用移液管吸取buffer布满整个器皿表面，确保细胞裂解完全。

将裂解产物转移至干净的1.5mlEP管中，然后进行第2步。

如果细胞是悬液培养，收集细胞（≤1,500rpm or 400*g）*5min，弃上清，加入TRK）裂解液，漩涡振荡混匀（具体略，见说明书）。

2．匀浆化裂解产物“裂解和匀浆化样品”－第四页有更详细的说明，如果细胞≤1*105，漩涡振荡1min。

样品匀浆化不完全会显著的减少RNA的产量并容易造成柱子堵塞。

a)用匀浆器（rotor-stator ）30 s，使样品匀浆化，而后进行第3步。

b)使裂解产物通过钝的针头的注射器（0.9mm直径20-gauge），使之匀浆化。

注射器要除RNase。

进行第3步。

c)将裂解物转移入omega homogenizer spin column,10000g离心1分钟。

将流出液移入新的tube中，进行第3步。

组织的步骤：1．TRK裂解缓冲液在离心管中裂解细胞，使用前每1ml TRK裂解缓冲液加入20ul的β－巯基乙醇。

350ulTRK裂解液最多能有效裂解20mg组织（-3mm3）。

对20mg以上组织用600ul的TRK裂解液。

然而，不要超过30mg组织，这是推荐的最大量。

OMEGA Plasmid Mini Kit I 中文说明书(官方原版)

D6942-00,D6943-00: 加入8ml无水乙醇 D6942-01,D6943-01: 加入80ml无水乙醇 D6842-02,D6943-02: 每瓶中加入80ml无水乙醇
离心操作方案 1. 将带有质粒的E.coli 接种于5ml LB/抗生素培养液中，37°C搖床培养12~16 h； 2. 取1.0~5.0ml的菌液，室温下10,000xg离心1min收集细菌。 3. 倒弃培养基。加入250ul Solution I/RNaseA混和液，漩涡振荡使细胞完全悬浮。 4. 往重悬混和液中加入250ul Solution II，轻轻颠倒混匀4-6 次。此操作避免剧烈混匀裂
洗脱:30-50ul Elution Buffer或无菌水
菌液用量最大结合力
1-5 mL
30 µg
5-15 mL
70 µg
15-50 mL 50-250 mL
250 µg 1.0 mg
货号和次数
D6942/3-00(5) D6942/3-01(50) D6942/3-02(200)
D6945-00(5) D6945-01(50) D6945-02(200)
E.Z.N.A.TM Plasmid Mini Kit I
Cat. No: D6942,D6943
快速流程图
重悬:250ul Solution I 裂解:250ul Solution II 中和:350ul Solution III
离心( 1 0 , 0 0 0 x g , 1 0 m i n )去除沉淀
解液且裂解反应不要超过5 min。 5. 加入350ul Solution III，温和颠倒数次至形成白色絮狀沉淀。 6. 室温下，≥10,000xg离心10min。 7. 转移上清液至套有2ml收集管的HiBind DNA结合柱中，室温下，10,000xg离心1 min，

无内毒素质粒大提取试剂盒中文操作指南

无内毒素质粒大提取试剂盒中文操作指南（omega）详细内容：E.Z.N.A. ® Fastfiler Endo-free Plasmid MaxiprepProtocol(无内毒素质粒大抽提)1. 在1-4升的培养瓶中加入200-500ml LB培养基,然后加入菌种于37℃摇床培养12-16小时；Tip:为获得最好的结果，请接种1ml培养过夜的菌种（12-16hr）。

并强烈推荐使用E.coli(endA-)品系用于常规的质粒提取，如DH5a和JM109。

注意：菌液的培养时间不能超过16小时；2. 收集200ml培养基至适当的离心管，室温下，3,500-5,000×g离心10分钟沉淀；3. 吸尽并去除培养基，用干净的吸水纸吸尽壁上多余的液体。

加12.0ml Solution I/RNase A到细菌培养物中，涡旋和枪头抽打细菌以重悬细胞；注：充分重悬细菌沉淀物对获得高产量的质粒是相当关键的。

充分重悬后溶液是均匀的，不存在小块物质；请尽量吸弃残余的培养基以防止稀释加入的溶液；4. 加入12.0ml SolutionII，轻轻颠倒旋转混匀7-10次至获得澄清的裂解液；室温放置3-5分钟可以有是必须的。

避免剧烈振荡混匀而打断染色体DNA，降低质粒的纯度。

（Solution II使用后请盖紧盖子且于室温保存）；5. 将取出Lysate Clearance Filter Syrine活塞，将其放置于架子上；6. 加入12 ml Neutralization Buffer，轻轻颠倒混匀几次至出现絮状沉淀。

这可能需要放置2-3分钟并间断颠倒混匀；7. 立即将裂解液转移到lysate Clearance Filter Syrine中，垂直放置5分钟。

这时白色的絮状沉淀物会漂上溶液的上层，裂解液可能开始流出过滤器，用新的50ml管子收集细菌裂解液，并将活塞轻轻插入过滤器中；8. 握住过滤器，轻轻推动活塞将裂解液打到收集管中；注意不要将任何杂质打到收集管中；9. 加入0.1体积的ETR Solution（蓝色）到收集液中，轻轻颠倒旋转混匀7-10次，冰浴放置20分钟；注意：加入ERT Solution后，溶液应变得浑浊，但冰浴静置后溶液应是澄清的。

RNA提取试剂盒说明书

Binding Capacity
Each HiBind ® RNA column can bind approximately 100 ìg RNA. Using greater than 250 mg fungal tissue in many cases will not drama tical improve yields and sometimes has
E.Z.N.A.™ Fungal RNA Protocol A (Standard Protocol)
Materials to be provided by user ! Microcentrifuge capable of 10,000 x g ! ! ! ! Nuclease-free microfuge tubes 2-mercaptoethanol Absolute (96%-100%) ethanol Isopropyl alcohol (isopropanol) Liquid nitrogen for freezing/disrupting samples Preheat an aliquot (100 ìl per sample) of DEPC-treated water at 65oC.
H iBind
®
R N A C olum ns
H om ogenizer C olum ns 2 m l C ollection Tubes B uffer R FL B uffer SP B uffer R B R N A W ash Buffer II R N A wash Buffer I D E P C W ater Instruction Booklet
Contents
Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Storage and Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Kit Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Before Starting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Fungal RNA Protocol A (Standard Protocol). . . . . . . . . . . . . . . . . . . . 4 Fungal RNA Protocol B (For difficult Samples)). . . . . . . . . . . . . . . . . . 6 On-Membrane DNase Digestion Protocol. . . . . . . . . . . . . . . . . . . . . . 8 RNA Isolation from Arthropods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Quantization of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Omega真菌RNA提取试剂盒R6840说明书(中文翻译版)

实验概要Omega真菌RNA提取试剂盒中文说明书(E.Z.N.A. Fungal RNA Kit)。

实验前准备高速离心机、无核酸酶的微量离心管（DEPC灭菌EP管）、β-巯基乙醇、无水乙醇、液氮（冷冻/破碎样品）、灭菌的研钵、65℃预热的DEPC水（每个样品100μl）。

实验前要用DEPC浸泡过夜并高压灭菌的东西有：蓝枪头、黄枪头、白枪头、5ml枪头及各枪头盒，小瓶（用来装乙醇等），EP管、玻棒、纱布（用来过滤菌丝）。

研钵用锡纸包裹180℃烘箱6h。

1. 取适量RB裂解液，按1ml裂解液加入20ul 2-疏基乙醇。

该混合液可于室温放置一周。

2. 用DEPC水配置一小瓶70%乙醇。

3. 按下表用无水乙醇稀释RNA Wash Buffer II，并于室温保存。

R6840-00: 加入8ml无水乙醇R6840-01: 加入48ml无水乙醇R6840-02: 每瓶加入48ml无水乙醇实验步骤1. 称取50-100mg经液氮研磨成粉末状的真菌样品至1. 5ml离心管，立即加入500ul RB/2-Me, 剧烈涡旋。

2. 转移液体至匀浆柱（试剂盒中提供的绿色柱子）中。

大于13,000×g离心5min。

3. 转移收集管中的上清(注意不要吸到沉淀)至新离心管，加入0. 5倍体积无水乙醇或，涡旋混匀15秒。

这一步如果发现有沉淀用注射器吹打或上下震荡10-15次。

(一般可转移450ul的上清液，可加入225ul 无水乙醇)。

这一步如果发现有沉淀用注射器吹打或上下震荡10-15次。

4. 把RNA柱套在新收集管，转移所有的混合液（即使有沉淀）至RNA柱子。

室温10,000×g离心30秒，弃去滤液。

如果出现堵柱现象，提高离心速度至14, 000xg。

(可选)膜上DNase消化:1) 加入300ul RNA Wash Buffer I至柱子中，按上柱条件离心，弃滤液；2) 配制DNase消化液( Digestion Buffer，73.5ul；RNase-Free DNase I，1.5ul)，混匀。

Omega公司RNA提取试剂盒说明书

RNA-Solv Reagent®RNA Isolation SolventWARNING: This reagent is toxic if swallowed and in contact with skin. Causes burns. After contact with skin, wash immediately with copious amounts of mild detergent and water. If you feel sick, seek medical advice at once and Quote UN2821.Product No:R6830-00 (5 ml)R6830-01 (100 ml)R6830-02 (200 ml)Storage Conditions:RNA-Solv is stable for at least 24 months®when stored at 2°C-8°C and yields reproducible results.IntroductionRNA-Solv® Reagent is a reagent system for the isolation of total RNA from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step RNA isolation method developed by Chomczynski and Sacchi (1).The sample is homogenized and lysed in RNA-Solv® Reagent which maintains the integrity of the RNA, while disrupting and denaturing endogenous RNases and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. RNA remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X10), and large quantities of tissue ( up to1 g) and cells 6(<10 ), of human, animal, plant, or bacterial origin. The simplicity 8of the RNA-Solv® Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA prepared in this manner 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 amplification by thermal cycling, treatment of the isolated RNA with RNase-free DNase I is recommended when the two amplimers lie within a single exon.Supplied By User•Chloroform (no isoamyl alcohol added)•Isopropyl alcohol•80% Ethanol (in DEPC-treated water)•RNase-free water•Tabletop centrifuge capable of 12,000 x g at room temperatureGeneral Notes Regarding RNase ContaminationWhenever working with RNA :•Always wear disposable gloves and change gloves frequently.•Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases.•In the presence of RNA-Solv® Reagent, RNA is protected from RNase contamination. Downstream sample handling requires that nondisposable glassware or plasticware be RNase-free.•Use only DECP-treated buffers. Add DEPC to a final concentration of 0.1%, incubate at 37C for 2 hours, andoautoclave at 121C. Do not add DEPC to Tris buffers. Suchobuffers must be prepared by using DECP-water.PrecautionUse only disposable polypropylene tubes for small samples and glass Corex tubes for larger samples. All tubes must be able to withstand 12,000 x g . Polystyrene tubes may crack with chloroform Before StartingA.Small Samples :To isolate RNA from very small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) of samples in 0.8 mL of RNA-Solv®, and add 1 mg RNase-free glycogen or yeast tRNA as carrier. This will improve yields obtained with precipitation.B.Difficult Animal Samples: Specimens containing large amounts of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and sperm, will require the following modification. After lysis/homogenization in RNA-Solv® Reagent, centrifuge at 12,000 x g for 10 minutes at room temperature to remove insoluble debris. Often a precipitate forms at the bottom of the tube, but with fatty tissue, a lipid layer will also form above the aqueous phase. The supernatant will contain the RNA and must be carefully transferred to a fresh 1.5 ml microfuge tube before proceeding.C. Interruption the procedure:Following lysis in RNA-Solv®Reagent and before addition of chloroform, samples can be stored at -70C for up to 3 months. In addition, once the RNA is oprecipitated in isopropanol, the pellet may be stored at -20C or -o70C for up to 1 year.oRNA-Solv Protocol for Total RNA Isolation®CAUTION: When working with RNA-Solv® Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20C-25C).o o1. Homogenization and lysis of samples: follow either method belowa) Tissue SamplesHomogenize tissue samples in 1 mL of RNA-Solv® Reagent per 50-100 mg of tissue using an appropriate mechanical homogenizer. Alternatively one can pulverize tissue in liquid nitrogen with mortar and pestle and transfer the powder to a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (Eppendorf, Cat No. 0030 120.973; VWR, Cat No. KT 749520-0000). The sample volume should not exceed 10% of the volume of RNA-Solv® Reagent used.b) Cells Grown in SuspensionPellet cells by centrifugation. Lyse cells in RNA-Solv® Reagent by repetitive pipetting. Use 1 mL of the reagent per 5-10 x 10of6 animal, plant or yeast cells, or per 1 x 10 bacterial cells. Washing8cells before addition of RNA-Solv® Reagent should be avoided as this increases the possibility of mRNA degradation and RNase contamination. For plant, fungal, and yeast cells mechanical or enzymatic homogenization may be required. Also, for plant, fungal, and yeast cells, we recommend the use of the E.Z.N.A.® Plant (R6627),Fungal (R6640), and Yeast (R6670) RNA Kits from Omega Bio-tek.c) Cells Grown in MonolayerLyse cells directly in a culture dish by adding 1 mL of RNA-Solv®Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a blue pipette tip. The amount of RNA-Solv®Reagent added is based on the area of the culture dish (~1 mL per 10 cm ). An insufficient amount of RNA-Solv® Reagent may result2in contamination of the isolated RNA with DNA. Always use more RNA-Solv® Reagent if in the lysate is too viscous to aspirate with a pipette.2. Add 0.2 mL of chloroform per 1 mL of RNA-Solv® Reagent. Cap sample tubes securely and vortex vigorously for 15 seconds. Incubate on ice for 10 minutes. This step is critical - do not change it.3. Centrifuge the samples at no more than 12,000 x g for 15 minutes 4E C.The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA remains entirely in the aqueous phase.4. Precipitation of RNA.Transfer no more than 80% of the aqueous phase to a fresh tube, and discard the lower organic phase. Precipitate the RNA from the aqueous phase by adding 500ìl of isopropyl alcohol per 1 mL of RNA-Solv® Reagent used for the initial homogenization. Incubate samples at room temperature 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes also at room temperature.Carbohydrate-rich samples: Plant samples of high polysaccharide content or animal tissues rich in glycosaminoglycans (proteoglycans) require the following modified precipitation method for obtaining pure RNA. Prepare Buffer A ( 1.2 M sodium chloride, 800 mM sodium citrate). Following step 3, add to the aqueous phase 0.3 ml isopropanol followed by 0.3 ml Buffer A per 1 ml RNA Solv ® Reagent used in step 1. Vortex to mix and centrifuge at no more than 12,000 x g for 10 minutes at room temperature. This high salt precipitation will reduce co-purification of complex carbohydrates.5. Wash RNA pellet. Discard the supernatant and wash the RNA pellet once with 1 ml 80% ethanol. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at room temperature.6.Reconstitute RNA. Carefully aspirate and discard the ethanol and briefly AIR DRY the RNA pellet for 2-5 minutes at room temperature. Do not use centrifugal devices equipped with a vacuum source as over-drying will lead to difficulty in re-dissolving RNA in water. Dissolve RNA in RNase-free water - a 5 minute incubation at 60 °C may be required. RNA can also be reconstituted in 100% formamide (deionized) and stored at -70°C.RNA is now suitable for RNase protection, northern analysis and reverse transcriptase reactions. For isolation of poly(A)+ RNA an additional ethanol precipitation is required. Add 1/8 X volume of RNase-free 3M NaAc, pH 6.0 followed by 2.5 X volume absolute ethanol. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge at 12,000 x g for 10 min at room temperature and discard the supernatant. Wash the pellet as before and reconstitute in DECP-treated water.Determination of Yield and QualityUV spectrophotometric analysis of the purified RNA is required for obtaining yield. To do so, dilute the RNA in an appropriate volume of TE buffer, pH 8.0 (not water; RNA yields low Abs ratio values if dissolved in acidic buffers) and measure absorbance at 260 nm and at 280 nm. RNA Conc = 40 ìg/ml X Dilution factor X Abs 260 nmTypical Abs 260 nm/ 280 nm ratios of 1.7-1.9 are obtained with the protocol. Yields vary depending of type and amount of starting material, and on condition of storage prior to processing. For assessing the quality of RNA, we recommend you perform denaturing agarose gel electrophoresis to confirm the integrity of purified material. Invariably, the full spectrum of RNAs, including 4S and 5S species are purified with RNA-Solv® Reagent.Expected Yields per 1 mg tissue or 10 cells:6Liver and spleen, 5-10 ìgKidney, 2-5 ìgBrain, 1-2ìgEndothelial cells, 7-12 ìgFibroblasts, 6-8ìgTroubleshooting•Low RNA Yields: Incomplete lysis of samples in RNA Solv Reagent. RNA pellet not completelt dissolved in DEPC-water.pH of diluent used for spectrophotometric analysis is too low.•Degraded RNA: Tissues were not immediately processed or frozen. Inadequate storage of starting material prio to isolation. Inadequate storage of RNA (-5 to -20°C, instead of -60 to -70°C) Trypsin/EDTA was used in dislodging monolayer cells. Buffers or plasticwasre were not RNase-free.Formaldehyde used for denaturing agarose-gel electrophoresis had a pH below 3.0.•Low Abs260/Abs280 ratios: Sample was diluted in water rather than TE. Acidic pH lowers absorbance ratios. Use TE buffer as diluent for readings. Insufficient RNASolv Reagent was used for lysis of sample. Ice incubation in step 2 was not performed. The aqueous phase was contaminated with the phenolic phase.•DNA contamination of RNA: Too little RNASolv Reagent used for sample processing causing inadequate separation of DNA/nucleoprotein complexes from aqueous RNA. The aqueous phase was contaminated with the phenol phase.Technical Support:Omega Bio-tek, USA - call toll-free : 1 888 832 8896References:1. Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156 (1987).2. Chomczynski, P. Biotechniques 15, 532 (1993).For laboratory research use only.CAUTION: Not for diagnostic use. The safety and efficacy ofthis product indiagnostic or other clinical uses has not been established.May,1999 (C). All rights reserved by Omega Bio-tek, Inc.RNA-Solv is a registered mark of Omega Bio-tek, Inc.。

OMG植物RNA提取试剂盒中文使用说明

OMG植物RNA提取试剂盒使用说明方案二：用于次生代谢物过多的植物组织1、液氮研磨，取100mg粉末至2ml离心管中，添加500ul Buffer RCL/β-巯基乙醇。

快速混匀，并保证无块状。

Note: 每1ml RCL Buffer中添加20ul β-巯基乙醇，混匀使用，混合液室温可以保存1周。

2、55℃水浴1-3min，室温最大转速（>14,000g）离心5min。

3、上清（大概可以得到450ul）转入含2ml收集管的gDNA Filter Colum中，室温14,000g离心2min。

4、加等体积Buffer RCB于收集管中，并上下颠倒5-10次混匀。

5、混合体系取1半置于含新2ml收集管的HiBind™RNA Mini Colum。

室温10,000g离心1min。

除去流动相，并把柱子放回收集管中。

6、把剩余的混合体系置于柱子中，室温10,000g离心1min。

除去流动相，柱子放回收集管中。

Note: 此时可选择DNase I处理步骤。

7、加400ul RWC Wash Buffer并室温10,000g离心1min，除去流动相和收集管。

8、把柱子放在一个新的2ml收集管上，加500ul RNA Wash Buffer II（用乙醇稀释过的），室温10,000g离心1min，除去流动相，柱子放回收集管。

Note: R6827-01，添加48ml无水乙醇于RNA Wash Buffer II的瓶中，并混匀后使用。

9、重复步骤8，除去流动相，清空收集管，把柱子放回收集管中，室温10,000g 离心2min。

10、RNA洗脱：把柱子置于新的1.5ml离心管上，加30-50ul DEPC水洗脱，确保水加在柱子中膜的正中央，室温静置洗脱2min，10,000g离心1min，1.5ml离心管中获得的液体即为RNA。

DNase I 处理1、当RNA全部吸附到HiBind™ RNA Mini柱上时，准备以下程序：A、加300ul RWC Wash Buffer到柱子上，10,000g离心1min。