omega DNA 提取试剂盒 说明书
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小提试剂盒提取质粒步骤(无内毒)
OMEGA小提试剂盒提取质粒步骤(中文翻译版)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500×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1000×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）。
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、disposable latex gloves。
细胞的步骤1．在microfuge tube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。
Pipette buffer over entire surface of vessel 以使裂解完全。
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flow-though移入新的tube中，进行第3步。
组织的步骤：1．TRK裂解缓冲液在microfuge tube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。
mobioDNA提取试剂盒说明书翻译
1.加2g土壤到15mL Bead Tube中2.加入0.25mLSR1和0.8mLSR2之后，加入2.5mL Bead Solution到Bead Tube3.加入3.5mL酚：氯仿：异戊醇25:24:1到试管中，加盖后涡旋混合直到分层消失。
4.最大转速涡旋震荡15min5.室温，2500g离心10min6.取出后，小心地转移上层水相到干净的15mL收集管中，弃掉下层酚7.加1.5mL SR3到水相中，然后涡旋混匀，4℃孵育10min8.室温，2500g离心10min。
转移上清到一个新的15mL收集管中9.加入5mL SR4溶液到装有上清的收集管中，混匀，室温孵育30min10.室温，2500g离心30min11.倒掉上清，将收集管倒置在纸上5min12.震荡SR5混合。
加1mL SR5到15mL收集管中，并反复吹打使完全重悬。
13.为每一个RNA样品准备一个RNA Capture ColumnA．拿去15mL收集管的盖子，将RNA Capture Column放入15mL收集管中。
B．加2mL SR5到RNA Capture Column中，让其完全流尽。
14.从第12步加入RNA样品至RNA Capture Column中，然后让其在重力作用下流尽。
收集液体。
15.用1mLSR5清洗柱子。
重力流尽，收集洗脱液。
16.转移柱子到新的收集管，加入SR6震荡混合，然后加入1mL SR6到RNA Capture Column洗脱RNA到15mL收集管中，重力流尽。
17.转移洗脱的RNA到2.2mL收集管中，并且加入1mL SR4.颠倒至少一次混合，然后-20℃孵育最少10min18.室温13000g离心15min浓缩RNA19.倒掉上清并倒转收集管在纸上10min晾干20.用100μL SR7溶液重悬RNA沉淀，去除其中的基因组。
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。
游离DNA提取试剂盒（磁珠法）说明书
游离DNA提取试剂盒（磁珠法）说明书【产品名称】通用名称：核酸提取或纯化试剂商品名称：游离DNA 提取试剂盒（磁珠法）英文名称：Magbind CFDNA Kit 该试剂盒提供了一种简单、快速、高效的游离DNA (Free circulating / Cell-free DNA)提取方法，适用于从血清、血浆、淋巴液、尿液等无细胞体液中提取游离DNA 。
纯化得到的离DNA 质量稳定、可靠，可用于下游常规实验。
该试剂盒可与转移液体法的核酸提取仪配套使用，简单、快速地进行大规模提取，大大降低了实验者的工作量和实验中的人为误差。
【预期用途】 样品裂解后，在高盐存在时，DNA 结合于硅基包被的磁珠表面，漂洗后，高纯度DNA 被洗脱于洗脱缓冲液或去离子水中。
DNA 得率与样品的类型、储存条件、时间以及个体间差异有很大关系。
【检验原理】50次/盒；400次/盒【包装规格】版本号：11/2020【样本要求】1．适用标本类型：新鲜或冷冻的全血（用柠檬酸盐、EDTA 或肝素等抗凝剂处理过的血液样品）、血浆、血清、淋巴细胞、无细胞体液等样本。
2．标本处理与保存：血清及血浆样本按常规方法制备，新鲜样本应尽快处理或分装后于-70℃冻存，避免反复冻融。
3．样本运输：应采用冰壶或者泡沫箱加冰或干冰密封运输。
【检验方法】实验前准备：向蛋白酶K 中加入指定用量的蛋白酶K 保存液使其溶解，终浓度为20 mg/ml ，-20℃保存。
1．向1.5 ml 离心管中加入20 μl 蛋白酶K 溶液。
2．向上一步的离心管中加入200 μl 血清/血浆样品。
注意：冻存样品需提前在4℃冰箱中放置使其溶化。
溶化过程中反复颠倒样品储存管使其混匀。
3．向上一步的离心管中加入200 μl 裂解缓冲液，涡旋振荡5秒钟使其充分混匀后将离心管放于56℃、1300 rpm 的Thermomixer 上振荡裂解10分钟。
注意：1）如无Thermomixer ，需将离心管放于56℃水浴锅或金属浴中孵育10分钟，期间每隔2分钟涡旋振荡5秒钟。
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、disposable latex gloves。
细胞的步骤1．在microfuge tube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。
Pipette buffer over entire surface of vessel 以使裂解完全。
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flow-though移入新的tube中，进行第3步。
组织的步骤：1．TRK裂解缓冲液在microfuge tube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。
omega质粒小提protocol
Omega 质粒小提试剂盒protocol主要试剂1. 使用前，将RNase A全部加入Solution I中并于4度保存。
2. 按下表用无水乙醇稀释DNA Wash Buffer，并于室温保存。
D6942-00: 加入6 ml无水乙醇D6942-01: 加入120 ml无水乙醇3. 按下表用异丙醇稀释HBC Buffer，并于室温保存。
D6942-00: 加入1.6 ml无水乙醇D6942-01: 加入16 ml无水乙醇D6942-02: 加入32 ml无水乙醇试剂配方及作用Solution I组分浓度25 mM Tris-HCl（pH8.0）,10 mM EDTA,50 mM 葡糖糖（Glucose）溶液1的主要作用是将菌体沉淀悬浮起来。
25mM Tris-HCl是缓冲溶液，保证反应体系的pH 恒定。
EDTA是金属离子螯合剂,10 mM EDTA的作用是与微生物体内的金属离子相互结合，抑制DNase的活性。
50 mM葡萄糖的作用是增加溶液的粘度，保证菌体悬浮，延缓菌体沉降的时间。
溶液1在使用前通常要加入RNA酶（RNase A）,RNA酶的作用很清楚，降解掉溶液中的RNA。
由于RNA酶属于蛋白质，蛋白质稳定性很差，所以加了RNase A的溶液1需要低温4℃保存。
Solution II组份浓度250 mM NaOH,1％（W/V）SDS（十二烷基硫酸钠）溶液2的主要作用是细胞裂解。
溶液1将细胞悬浮后,就需要添加溶液2了,溶液2的作用就是对细胞进行裂解。
溶液2只包括两种成分：氢氧化钠和SDS。
真正起到到细胞裂解作用的是氢氧化钠,这也是为什么这个方法会被叫做碱裂解法。
氢氧化钠会破坏细胞膜的结构,使之发生bilayer（双层膜）结构向micelle（微囊）结构的相变化,从而导致细胞的裂解。
SDS的作用将在下一步提到。
添加溶液2后需要注意的一个是时间一定不能过长,另一个是不可以剧烈震动离心管。
时间过长以及剧烈震动都将导致氢氧化钠破坏基因组DNA,断裂的基因组DNA碎片将会与相似大小质粒一同被抽提,污染样品。
Omega真菌DNA提取试剂盒说明书
Introduction
The E.Z.N.A.™ High Performance (HP) Plant DNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from fresh and dried plant tissue samples rich in polysaccharides or lower DNA contents. Up to 100 mg of wet tissue (or 30 mg dry tissue) can be processed in less than 1 hour. The system combines the reversible nucleic acid-binding properties of the HiBind® matrix with the speed and versatility of spin column technology to eliminate polysaccharides, phenolic compounds, and enzyme inhibitors from plant tissue lysates. Purified DNA is suitable for PCR, restriction digestion, and hybridization techniques. There are no organic extractions thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.
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。
试剂盒提DNA步骤
OMEGA--D5525-01型水样中DNA提取试剂盒E．Z．N．A TM WaterDNA KitD5525-01试剂盒内含：Hinbind DNA结合柱50个，2mL收集管100个，玻璃珠27g，HTR试剂10mL，BufferSLX 180mL，SP2 Buffer 60mL,XP1 Buffer40mL,Elution Buffer 30mL,DNA Wash Buffer 20mL。
有关Buffer GPS硅胶柱（即Hinbind DNA结合柱）经Buffer GPS预处理后可大大减少柱子中硅胶膜的憎水基团，提高核酸的结合能力。
柱子在长期放置过程中会同空气中的电荷/尘埃发生反应而影响其核酸的结合能力。
Buffer GPS是强碱性溶液，若不小心碰到，请用大量自来水清洗。
用完后需盖紧瓶盖，以免接触空气。
室温保存Buffer GPS。
在保存过程中可能会有沉淀生成，请加热至37℃使沉淀完全消失。
需要准备的：预定工作台(9：00—16:00)超净台在开紫外前先用75%的酒精棉擦拭以使台面尽量无菌，预定4℃离心机(10:00-（至少定3h），采水样，清洗抽滤装置（擦洗灭菌），（95%乙醇泡烧）无菌镊子、剪刀，水浴锅（先设定为70℃，冰盒（70℃水浴时制即可）、试剂盒，异丙醇，无菌锡箔纸、吸水纸、1000μL移液器及枪头，黄枪头，计时器，灭菌勺，离心管架，转速约14000g的离心机，灭菌50mL离心管，灭菌1.5mL离心管，ddH2O，0.22μm微孔滤膜，75%酒精及灭菌脱脂棉（用来擦洗已提过水样的漏斗），放镊子剪刀大离心管等的试剂架。
Buffer GPS对硅胶柱的前处理：（在超净台中操作，可以在第10步时处理柱子）1.取一个新的Hinbind DNA结合柱装在收集管中，吸取合适体积的Buffer GPS平衡缓冲液（小柱提取用200μL，中柱提取用1mL，大量提取用3mL）至柱子中。
室温放置3~5min。
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、disposable latex gloves。
细胞的步骤1．在microfuge tube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。
Pipette buffer over entire surface of vessel 以使裂解完全。
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flow-though移入新的tube中，进行第3步。
组织的步骤：1．TRK裂解缓冲液在microfuge tube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。
omega真菌DNA提取试剂盒说明书
!
Pre-heat Elution Buffer to 65E C.
!
Dilute Wash Buffer Concentrate with ethanol as follows and store at room temperature.
D3390-00 D3390-01
Add 18 mL absolute (96%-100%) ethanol. Add 72 mL absolute (96%-100%) ethanol to each bottle. Add 90 mL absolute (96%-100%) ethanol to each bottle.
E.Z.N.A . Fungal DNA Kit 1 1 /2 0 0 7
2
E.Z.N.A . Fungal DNA Kit 1 1 /2 0 0 7
Kit Contents
Product N um ber H iBind
®
D 3390-00 5 10 5 ml 2 mL 2 mL 1.5 m L 40
For processing #50 m g powdered tissue. Yield is sufficient for several tracks on Southern assay. For processing #200 m g fresh (or frozen) tissue. Yield is sim ilar to A .
Introduction Contents
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Materials to Be Provided by User ! Tabletop microcentrifuge and nuclease-free 1.5 ml tubes ! Water bath set to 30oC ! Shaking water bath set to 55oC ! Incubator or waterbath set to 65oC ! Absolute ethanol (96%-100%) - Do not use other alcohols
Add 8 ml absolute (96%-100%) ethanol Add 80 ml absolute (96%-100%) ethanol Add 80 ml absolute (96%-100%) ethanol per bottle
Store the diluted DNA Wash Buffer at room temperature.
乙醇
Page 3 of 8
Before Starting
! Please read the entire booklet to become familiar with the E.Z.N.A.® Bacterial DNA Kit procedure.
! Prepare a lysozyme stock solution at 50 mg/ml and aliquot into adequate portions. Store each aliquot at -20oC and thaw before use. Each sample will require 20 ìl of this solution.
Kit Contents
Product Purification times HiBind® DNA Mini Columns 2 ml Collection Tubes Buffer BTL Buffer BDL Buffer HB DNA Wash Buffer concentrate Glass Powder Lysozyme Elution Buffer Proteinase K RNase A User Manual
! Equilibrate Elution Buffer provided to 65oC.
! Dilute DNA Wash Buffer Concentrate with ethanol as follows and store at room temperature:
D3350-00 D3350-01 D3350-02
Binding Capacity
Each HiBind® DNA column can bind approximately 100 ìg genomic DNA. Using greater than 1 x 109 bacterial cells is not recommended.
Page 2 of 8
Grow Bacteria in LB media to log phase. (Overnight culture can be used in many cases.)
2. Harvest no more than 3 ml culture by centrifugation at 4,000 x g for 10 min at room temperature.
! Carry out all of centrifugation step at room temperature.
Page 4 of 8
E.Z.N.A.TM Bacterial DNA Spin Protocol 1. This method allows genomic bacterial isolation from up to 3 ml LB 0 Preps
200 400 50 ml 50 ml 110 ml 3 x 20 ml 8.0 g 4 x 50mg 40 ml 4 x 1.4 ml 1.1 ml
1
Buffer BDL contains a chaotropic salt. Use gloves and protective eyeware when handling this solution.
Introduction
The E.Z.N.A.® Bacterial DNA Kit allows rapid and reliable isolation of high-quality total cellular DNA from a wide variety of bacterial species. Up to 1 x 109 bacterial cells can be processed. The system combines the reversible nucleic acid-binding properties of Omega Bio-Tek’s HiBind® matrix with the speed and versatility of spin column technology to yield up to approximately 15-30 ìg of DNA with an A260/A280 ratio of 1.7-1.9. Purified DNA is suitable for PCR, restriction digestion, and hybridization techniques. There are no organic extractions, thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.
D3350-00 5 Preps 5 10 1.5 ml 2 ml 3 ml 2 ml 200 mg 5 mg 1 ml 110 ìl 30 ìl 1
D3350-01 50 Preps
50 100 20 ml 20 ml 30 ml 20 ml 2.0 g 50 mg 10 ml 1.4 ml 275 ìl
D3350-00 D3350-01 D3350-02
dissolve with 100 ìl of Elution Buffer dissolve with 1 ml of Elution Buffer dissolve with 1 ml of Elution Buffer for each tube
N O TE: E .Z.N.A.® B acterial D N A K it w ill isolate all cellular D N A, including plasm id D N A.
Overview
If using the E.Z.N.A.® Bacterial DNA Kit for the first time, please read this booklet to become familiar with the procedures. Bacterial cells are grown to log-phase and harvested. The bacterial cell wall is removed by lysozyme digestion, followed by Proteinase K digestion. Following lysis, binding conditions are adjusted and the sample is applied to a HiBind® DNA spin-column. Two rapid wash steps remove trace salts and protein contaminants, and finally DNA is eluted in water or low ionic strength buffer. Purified DNA can be directly used in downstream applications without the need for further purification.
Storage and Stability
All components of the E.Z.N.A.® Bacterial DNA Kit, except the RNase A and Lysozyme can be stored at 22EC-25EC and are guaranteed for at least 24 months from the date of purchase. Once reconstituted in water, lysozyme must be stored at -20EC. Store RNase A at -20EC. Proteinase K should be stored at 15EC - 25EC. Under cool ambient conditions, a precipitate may form in the Buffer BDL/BTL. In case of such an event, heat the bottle at 37EC to dissolve. Store Buffer BDL/BTL at room temperature.
Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Storage and Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Binding Capacity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Kit Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Materials to Be Provided by User.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Before Starting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 E.Z.N.A.TM Bacterial DNA Spin Protocol.. . . . . . . . . . . . . . . . . . . . . . . . . . 5 E.Z.N.A.TM Bacterial DNA Vacuum/Spin Protocol.. . . . . . . . . . . . . . . . . . . 7 Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8