天根DP117-无内毒素质粒大提试剂盒说明书精编版

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内毒素去除试剂盒说明书

内毒素去除试剂盒说明书I. DESCRIPTION细菌内毒素(脂多糖，LPS)是革兰氏阴性细菌细胞壁的主要成分。

人们在研究革兰氏阴性菌血症和内毒素血症的过程中，发现细菌内毒素在其中起着关键的作用，内毒素使机体免疫功能严重受损，进一步的发展可能引发脓毒性休克，弥散性血管内凝血，急性呼吸窘迫综合症，全身炎症反应综合症或致命性多器官功能衰竭。

因此，内毒素的去除对于人类疾病治疗的注射型药品、生物制品等都是必须的，尤其对于基因药物、蛋白药物、单抗克隆药物、治疗性疫苗、小分子化学药物等生物制品的下游纯化处理来说就显得非常重要。

本试剂盒使用的是一类高效内毒素去除树脂，它以修饰过的多粘菌素B（PMB）为配体，进行特异性的内毒素去除。

经此亲和树脂纯化，样品最终的内毒素水平可低于0.1 EU/ml。

亲和树脂的主要特性如下表：规格 1.5 ml预装柱结合能力高达2,000,000 EU/ml 树脂配基修饰的多粘菌素B (PMB)pH 范围pH 5-10基质4% 交联的琼脂糖凝胶颗粒大小90 µm保存温度2°C-8°C使用寿命18个月平衡缓冲液磷酸盐缓冲液，pH 8.0适合纯化对象蛋白，多肽，抗体，多糖等离子条件0.1-0.5 M NaCl可耐受试剂20% DMSO, 20% 乙醇, 20% 甘油;1 M 尿素, 300 mM 咪唑; 0.05% 吐温-20, 10 mM DTT 等II. KEY FEATURES·高稳定性，高去除效率·高结合力：> 2, 000, 000 EU / ml (CV)·无需恒流泵即可调节流速·重复使用5次不会改变柱子效果·使用方便，试剂盒中提供无热源缓冲液，无热源收集管，无热源枪头等III. CONTENTS试剂盒内容 3 – 5个测试亲和树脂 1.5 ml 预装柱再生缓冲液125 ml平衡缓冲液125 ml流速控制器1个无热源接收管 1 包(3个/包)无热源枪头(1 ml) 2包(6个/包) 说明书1份IV. MATERIALS AND EQUIPMENT NEEDED BUT NOT PROVIDED1. 铁架台2. 0.1 M的氢氧化钠和0.1M盐酸, 用于调节样品pH值V. ENDOTOXIN REMOVAL PROTOCOL样品处理：样品的pH值和离子强度在内毒素去除的过程中起着重要作用。

大型大量质粒提取试剂盒操作方法及步骤说明书

杭州昊鑫生物科技股份有限公司 htpp://PhasePrep EndoFree Maxi Kit大型/大量质粒提取试剂盒目录号：PL1401适用范围：适用于大量高纯或者转染级质粒制备和BAC/PAC大型质粒制备试剂盒组成、储存、稳定性：试剂盒组成保存20次（PL1401）RNaseA（10mg/ml）-20℃ 1.3ml溶液P1 4℃130ml溶液P2 室温100 ml溶液PⅢ室温110 ml杂质清除剂A 室温 3 ml杂质清除剂B 室温30 ml内毒素清除剂-20℃10 ml本试剂盒在室温储存18个月不影响使用效果。

储存事项:1.第一次使用时，将试剂盒所带全部的RNase A加入溶液P1后（终浓度100μg /ml）置于4℃保存。

如果溶液P1中RNase A失活，提取的质粒可能会混杂有微量RNA 残留，在溶液P1中补加RNase A即可。

2.内毒素清除剂在4℃可保存一个月，如果要长期保存，建议保存在－20℃!3.环境温度低时溶液P2中SDS可能会析出出现浑浊或者沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。

4.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。

产品介绍：本试剂盒用碱裂解法从培养菌中提取质粒DNA，采用独特的溶液配方和内毒素清除试剂，只需要几次简单离心去除蛋白质、多糖、内毒素、RNA等杂质，获得高质量的质粒DNA。

纯化DNA的OD260/280通常在1.8左右，得到的质粒可直接应用于细胞转染甚至动物体内实验等对DNA纯度要求很高的工作中。

纯化后期过程均在1.5ml 小离心管中操作，方法简单，不需特殊设备，无需过柱，不用酚氯仿抽提；基本可完全回收细菌裂解释放出的质粒，不必担心质粒DNA的丢失。

本方法提取纯化质粒DNA，对质粒损伤小，即使是10kb甚至100kb以上的大型质粒或超大型BAC/PAC质粒，只要碱裂解法能够提取，就可以有效纯化。

快速型无内毒素质粒超大量 3 提取试剂盒手册说明书

EZgene TM EndoFree plasmid ezFlow ezFilter Megaprep3kit(BG0060) HandbookFor research use only.Not intended for diagnostic testing.ContentsContents (1)Introduction (2)Important Notes (2)Storage and Stability (3)BeforeStarting (4)Safety Information (4)Kit Contents (5)EZgene TM Plasmid ezFilterMegaprep3Protocol (6)Purification of Low-Copy-Number Plasmid and Cosmid (8)快速型无内毒素质粒超大量3提取试剂盒简明步骤 (9)Trouble ShootingGuide (12)IntroductionKey to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBind TM matrix while proteins and other contaminates are removed under certain optimal conditions.Nucleic acids are easily eluted with sterile water or elution buffer.Unlike other procedures,our patented plasmid purification kit has no guanidine salt in the buffer,the purified DNA is guanidine/ion exchange resin residues free which enable the high performance of downstream applications such as transfection, restriction mapping,library screening,sequencing,as well as gene therapy and genetic vaccinations.Important Notesumbers::The yield of plasmid DNA depends on the origin of the Plasmid Copy N umbersreplication and the size of the plasmid.The protocols are optimized for high copy number plasmid purification.For low copy number plasmids,both the culture volume and the buffer volume need to be scaled up2times.Reference Table1for the commonly used plasmids,Host Strains:The strains used for propagating plasmid have significant influence on yield.Host strains such as Top10and DH5a yield high-quality plasmid DNA. endA+strains such as JM101,JM110,HB101,TG1and their derivatives,normally have low plasmid yield due to either endogenous endonucleases or high carbohydrates released during lysis.We recommend transform plasmid to an endA-strain if the yield is not satisfactory.Please reference Table2for the endA information.Table2endA strains of E.Coli .Optimal Cell Mass (OD 600x mL of Culture):This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani)for 12-16hours to a density of OD 6002.0to 3.0.If rich medium such as TB or 2xYT are used,make sure the cell density doesn’t exceed 3.0(OD 600).A high ratio of biomass over lysis buffers result in low DNA yield and purity.Culture Volume Volume::Use a flask or tube with a volume at 4times the culture medium to secure optimal condition for bacteria growth.Don’t exceed the maximum culture volume suggested in the protocol.Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity.Table 3The optimal cell mass mass,,culture Volumeand Binding Capacity for the mega DNA units,S torage and StabilityBuffer A1should be stored at 4°C once RNase A is added and Buffer ER should be stored at 4°C.All other materials can be stored at room temperature (22-25o C).The Guaranteed shelf life is 12months from the date of purchase.Before StartingPrepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps.Important:I.RNase A:It is stable for more than half a year when stored at roomtemperature.Spin down RNaseA vial briefly.Add the RNaseA solution to Buffer A1and mix well before use.II.Buffer ER should be stored at4°C.III.Buffer B1precipitates below room temperature.It is critical to warm up the buffer at50°C to dissolve the precipitates before use. IV.Buffer N3may form precipitates below10°C,warm up at37°C to dissolve the precipitates before use.V.Keep the cap tightly closed for Buffer B1after use.VI.Make sure the availability of centrifuge and vacuum manifold, especially,after mixing the lysate with ethanol,the sample needs to be processed immediately by vacuum.Materials supplied by users:VII.100%ethanol.VIII.Pump-driven vacuum system,1,000mL bottle(Corning#430518 or430282)or equivalent pyrex glass bottles.IX.50mL conical tubes.X.High speed centrifuge tube for endotoxin removal if desired. Safety Information�Buffer N3contains acetic acid,use gloves and protective eyewear when handling.�Buffer N3,Buffer RET contains chaotropic salts,which may form reactive compounds when combines with bleach.Do not add bleach or acidic solutions directly to the preparation waste.Kit Contents*Add216mL(BG0059)or320mL(BG0060)96-100%ethanol to each DNA Wash Buffer bottle before use.EZgene TM Plasmid ezFilter ezFilterMegaprep Megaprep 3Protocol I.Inoculate 600600mLmL LB containing appropriate antibiotic with 500µL fresh starter culture.Grow at 37°C for 14-16h with vigorous shaking.Note :The best way to prepare a starter culture:Inoculate a single colony from a freshly grown selective plate into 1mL LB medium containing the appropriate antibiotic and grow at 37°C for 6-8h with vigorous shaking (~250rpm).The buffer volumes need to be scaled up if processing over 500mL of culture.II.Harvest 600mL overnight bacterial cells by centrifugation at 5,000x g for 10minutes at room temperature.Decant or aspirate medium and discard.Note :Remove the residual medium completely for optimal cell lysis and neutralization.III.Resuspend the bacterial pellet in 30mL Buffer A1(Add RNase A to BufferA1before use).Pipet or vortex till thebacterial pellet dispersed thoroughly(Complete resuspension is critical for optimal yields).Then add 1.5mL Bu Bufferffer ER into the suspended bacterial culture.Mix well by inverting 5-10times.Note :if room temperature is below 25°C,warm up the mix solution after adding Buffer ER at 45°C for 5min.IV.Add 27mL Buffer B1.Mix gently but thoroughly by inverting 10times and incubate atroom temperature for 5minutestoobtain a cleared lysate.Do not incubate longer than 5minutes.Then add 3mL Buffer D1,mix gently and incubating for another 5minutes.Over-incubating causes genomic DNA contamination and plasmid damage .Avoid vigorous mixing as this will shear the genomic DNA.V.Add 8mL Buffer N3and mix immediately by inverting 5times till a flocculentwhite precipitate forms.Vortex the lysate for 5seconds.Note:It is critical to mix the lysate well,if the mixture still appears conglobated,brownish or viscous,more mix is required to completely neutralize the solution.VI.Attach the 2-layer filter unit to a sterile 500mL or 1000mL standard bottle(Corning#430518or 430282or equivalent pyrex glass bottle)and screw tight.Connect the unit to a pump-driven vacuum system.VII.Transfer the clear lysate from the bottom of the mixture (use a 50mL serological pipet)to the filter unit.Stand by for 5minutes and turn on the vacuum with low vacuum force and increase to maximum vacuum force after 5minutes.Figure1.Instruction of filter assembling.Note11:If the flow through gets too slow,turn off the vacuum and wait for1minute.NoteCarefully detach the upper filter cup and replace it with the replacement cup.Assemble the unit as Figure1.Pour the lysate from the original cup to the replacement cup.Turn on the vacuum and filter the rest of the lysateN ote2:Low vacuum force prevents clogging of the filter membranes.Note3:Use a50mL serological pipet to transfer the relatively clear lysate from the bottom of the lysate bottle to the filter unit.This will speed up the flow rate of the filter unit.Normally around200mL lysate can be filtered through the filter unit within20-30 minutes.Pour the remaining white precipitates to the filter unit when most of the lysate has been filtered through.VIII.When most of the lysate has been filtered through the unit,turn off the vacuum,wait for1minute,detach the unit and discard the upper filter cup including the rubber rings.Note:The DNA is in the collection bottle.IX.Connect the DNA unit to a500mL or1,000mL standard bottle and screw tight.Connect the DNA unit to the vacuum with the vacuum off.Add1volume of Buffer RET(For example,60m L of Buffer RET to60m L of clear lysate), and add36mL100%ethanol to the lysate bottle.Mix well by sharp hand shaking3-5times and immediately pour half of the lysate/ethanol mixture to the DNA unit and turn on the vacuum.X.Pour the rest of the lysate/ethanol mixture into the DNA unit.When all the lysate pass through the DNA unit,vacuum for1minute.XI.Wash the DNA membrane with50mL DNA Washing Buffer and vacuum for 1minute at maximum force.Wash the DNA membrane with another50 mL DNA Washing Buffer and vacuum for1minute at maximum force. XII.Add50mL100%ethanol evenly to the DNA membrane and vacuum for1minute.Turn off the vacuum,wait for1minute,and discard the liquid waste in the bottle.Reconnect the bottle to the DNA binding unit.Turn on the vacuum for10minutes at maximum force(It is critical to dry the residual ethanol for optimal yield).Turn off the vacuum,incubate at65o C for10min will help to remove the ethonal and increase the elution efficiencyXIII.Wait for1minute,and replace the500mL or1,000mL standard bottle with a sterile50mL conical tube,screw tight.XIV.Add8mL Endofree Elution Buffer evenly to the membrane and incubate for5minutes.Turn on vacuum to elute DNA.Typically3~5mL of solution can be collected.This is the1st elution.XV.Turn off the vacuum and replace the50mL conical tube with another sterile 50mL conical tube,screw tight.Add3mL Endofree Elution Buffer and incubate for3minute.Turn on the vacuum and collect the2nd elution,typically mL of solution can be collected.1~21~2mLNote:The DNA is ready for downstream applications such as transfection of endotoxin-sensitive cell lines,primary cultured cells or microinjection.Note:Two elutions give rise to maximum DNA yield.For maximum yield and higher concentration,pool the elutions together,add0.1volume3M Potassium Acetate or Sodium acetate(pH5.2)and0.7volume isopropanol.Centrifuge at top speed for10 min.Discard supernatant.Wash the DNA with1000µL70%ethanol,centrifuge for5 min,carefully decant.Air-dry the pellet for10-20minutes in a tissue culture hood.Resuspend the DNA in Endofree Elution Buffer.Note:Use lessEndofree Elution Buffer if high concentration is desired.g/mL L)=OD260nm x50x dilution factor.DNA concentration(µg/mPurification of Low-Copy-Number Plasmid and CosmidThe yield of low copy number plasmid is normally around0.1–1µg/mL of overnight culture.For isolating low copy number or medium copy number plasmid DNA,use the following guideline:•Culture volume:Use2x volume of the high copy number cultureBuffer B1,Buffer D1and Buffer N3•Use2x volume of the Buffer A1,Buffer ER,ER,Bufferand100%ethanol.Additional buffers can be purchased from Abgent.•use same volume of DNA Wash Buffer and Endofree Elution BuffeBuffer.r.快速型无内毒素质粒超大量3提取试剂盒简明步骤（详细内容请参考说明书英文部分）�实验前准备RNase A:常温下可稳定贮藏半年，使用前将提供的所有RNase A瞬时离心后加入Buffer A1,使用后将Buffer A1/RNase A置于4o C保存。

无内毒素质粒大提取试剂盒中文操作指南

详细内容：. ® (无内毒素质粒大抽提). 在升地培养瓶中加入培养基,然后加入菌种于℃摇床培养小时；文档收集自网络，仅用于个人学习:为获得最好地结果，请接种培养过夜地菌种（）.并强烈推荐使用()品系用于常规地质粒提取，如和.注意：菌液地培养时间不能超过小时；文档收集自网络，仅用于个人学习. 收集培养基至适当地离心管，室温下，×离心分钟沉淀；. 吸尽并去除培养基，用干净地吸水纸吸尽壁上多余地液体.加到细菌培养物中，涡旋和枪头抽打细菌以重悬细胞；文档收集自网络，仅用于个人学习注：充分重悬细菌沉淀物对获得高产量地质粒是相当关键地.充分重悬后溶液是均匀地，不存在小块物质；请尽量吸弃残余地培养基以防止稀释加入地溶液；文档收集自网络，仅用于个人学习. 加入，轻轻颠倒旋转混匀次至获得澄清地裂解液；室温放置分钟可以有是必须地.避免剧烈振荡混匀而打断染色体，降低质粒地纯度.（使用后请盖紧盖子且于室温保存）；文档收集自网络，仅用于个人学习. 将取出活塞，将其放置于架子上；. 加入，轻轻颠倒混匀几次至出现絮状沉淀.这可能需要放置分钟并间断颠倒混匀；文档收集自网络，仅用于个人学习. 立即将裂解液转移到中，垂直放置分钟.这时白色地絮状沉淀物会漂上溶液地上层，裂解液可能开始流出过滤器，用新地管子收集细菌裂解液，并将活塞轻轻插入过滤器中；文档收集自网络，仅用于个人学习. 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；. 加入体积地（蓝色）到收集液中，轻轻颠倒旋转混匀次，冰浴放置分钟；注意：加入后，溶液应变得浑浊，但冰浴静置后溶液应是澄清地.文档收集自网络，仅用于个人学习. ℃孵育分钟，溶液重新变为浑浊.室温下，×离心分钟，（蓝色）分层于离心管底部.文档收集自网络，仅用于个人学习. 把上面地水相（上清液）转移到新地离心管中，加入体积地，轻轻颠倒旋转混匀次.注意：转移上清液时不要吸到任何溶液，因为其含有高浓度地内毒素；文档收集自网络，仅用于个人学习. 平衡柱子：用套在收集管中，加入至柱子中，室温下×离心分钟；去除滤过液并重复使用收集管；文档收集自网络，仅用于个人学习. 加入澄清地裂解液至柱子中，×离心分钟.去除滤过液并重新收集管；. 将剩余地裂解液加到柱子上，按上述条件离心，去除滤过液并重新使用收集管；. 加入至柱子中，×离心分钟，去除滤过液并重新使用收集管；文档收集自网络，仅用于个人学习. 加入(已用乙醇稀释)至柱子中，×离心分钟，去除滤过液并重新使用收集管；文档收集自网络，仅用于个人学习. 加入至柱子中，×离心分钟，去除滤过液和收集管；文档收集自网络，仅用于个人学习. 将柱子套在新地离心管中，加入或水至柱子地基质.室温下×离心洗脱.文档收集自网络，仅用于个人学习. ® ( )文档收集自网络，仅用于个人学习. 按步骤准备澄清地裂解液；. 平衡柱子：将同真空装置相连接，加入至柱子中，提供真空分钟至裂解液完全滤过膜；文档收集自网络，仅用于个人学习. 将澄清地裂解液至柱子中，提供真空让所有地溶液滤出柱子；加入至柱子，提供真空吸尽液体；文档收集自网络，仅用于个人学习. 加入（已经用无水乙醇稀释）至柱子中，提供真空让溶液滤出柱子；文档收集自网络，仅用于个人学习. 重用至柱子中，提供真空让溶液滤出柱子；溶液滤出柱子后，将柱子转移至收集管中，另外提供真空分钟，室温下，×离心分钟以洗脱.第二次洗脱可能是需要地；文档收集自网络，仅用于个人学习. 去除柱子，然后将产物保存于℃.。

质粒大提说明书

Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TM280Revised 11/09Page 11.Description (1)2.Product Components and Storage Conditions ............................................23.Equipment, Supplies and Preparation of Solutions .. (3)4.PureYield™ Plasmid Maxiprep System Protocol (4)A.Preparation and Lysis of Bacterial Cell Cultures (4)B.DNA Purification..................................................................................................55.Supplemental Information (9)A.Considerations Regarding the PureYield™ Plasmid MaxiprepSystem (9)B.Selection and Preparation of Plasmids and E. coli Strains (10)C.Choosing a Bacterial Strain (10)position of Buffers and Solutions (11)6.Troubleshooting (12)ing the Vac-Man ®Laboratory Vacuum Manifold (14)A.Description (14)B.Setup and Operation (14)8.Related Products (16)1.DescriptionAs research moves from DNA sequencing to analysis of gene function, the need has increased for rapid methods by which to isolate large quantities of high-quality plasmid DNA. The PureYield™ Plasmid Maxiprep System (a)is designed to isolate high-quality plasmid DNA for use in eukaryotic transfection and in cell-free expression experiments. The system provides a rapid method for purification using a silica-membrane column. Plasmid DNA can be purified in approximately 60 minutes, greatly reducing the time spent on purification compared to silica-resin or other membrane column methods.The PureYield™ Plasmid Maxiprep System also incorporates a unique Endotoxin Removal Wash designed to remove substantial amounts of protein,RNA and endotoxin contaminants from purified plasmid DNA, and improve PureYield™ Plasmid Maxiprep System All technical literature is available on the Internet at: /tbs/ Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on useof this system. E-mail: techserv@the robustness of sensitive applications such as eukaryotic transfection, in vitrotranscription and cell-free expression. Purification is achieved withoutisopropanol precipitation of purified plasmid DNA, providing rapidpurification as well as a high concentration of pure plasmid DNA. ThePureYield™ Plasmid Maxiprep System purifies up to 1mg of plasmid DNAwith an A260/A280>1.7 from 250ml of overnight bacterial culture, transformedwith a high-copy-number plasmid.The PureYield™ System requires a vacuum pump and manifold (e.g., the Vac-Man®Laboratory Vacuum Manifold, 20-sample [Cat.# A7231]), a centrifugewith a fixed-angle rotor for lysate clearing and a tabletop centrifuge with aswinging bucket rotor for elution by centrifugation. Alternatively and foroptimal DNA recovery and yield, elution can be performed by vacuum, withthe Eluator™ Vacuum Elution Device (Cat.# A1071; see Section 4.B). Use of theEluator™ Elution Device eliminates the need for a tabletop centrifuge andswinging bucket rotor.2.Product Components and Storage ConditionsProduct Size Cat.# PureYield™ Plasmid Maxiprep System 2 preps A2391 Each system contains sufficient reagents for 2 × 250ml preps. Includes:•32ml Cell Resuspension Solution (CRA)•32ml Cell Lysis Solution (CLA)•30ml Neutralization Solution (NSB)•8.1ml Endotoxin Removal Wash•18ml Column Wash• 3.75ml Nuclease-Free Water• 2 each PureYield™ Clearing Columns• 2 each PureYield™ Maxi Binding ColumnsProduct Size Cat.# PureYield™ Plasmid Maxiprep System10 preps A2392 Each system contains sufficient reagents for 10 × 250ml preps. Includes:•125ml Cell Resuspension Solution (CRA)•125ml Cell Lysis Solution (CLA)•130ml Neutralization Solution (NSB)•36.5ml Endotoxin Removal Wash•82ml Column Wash•25ml Nuclease-Free Water•10 each PureYield™ Clearing Columns•10 each PureYield™ Maxi Binding ColumnsPromega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TM280Printed in USA. Page 2Revised 11/09Product Size Cat.# PureYield™ Plasmid Maxiprep System25 preps A2393 Each system contains sufficient reagents for 25 × 250ml preps. Includes:•315ml Cell Resuspension Solution (CRA)•315ml Cell Lysis Solution (CLA)•315ml Neutralization Solution (NSB)•85.3ml Endotoxin Removal Wash•210ml Column Wash•50ml Nuclease-Free Water•25 each PureYield™ Clearing Columns•25 each PureYield™ Maxi Binding ColumnsStorage Conditions:Store all system components at room temperature(22–25°C).Available SeparatelyProduct Size Cat.# Cell Resuspension Solution (CRA)315ml A7115 Cell Lysis Solution (CLA)315ml A7125 Eluator™ Vacuum Elution Device* 4 each A1071 Neutralization Solution (NSB)500ml A1485 Vac-Man®Laboratory Vacuum Manifold, 20-sample each A7231 *For Laboratory Use.3.Equipment, Supplies and Preparation of SolutionsMaterials to Be Supplied by the User(Solution compositions are provided in Section5.D.)•isopropanol•ethanol, 95%•tabletop centrifuge•swinging bucket rotor•disposable plastic 50ml screw-cap tubes (Corning Cat.# C4558 orBD Falcon™ Cat.# 352070)•vacuum pump, single- or double-stage,producing approximately 650mm Hg(25.6 inches Hg) of pressure (see tablefor pressure comparisons)•fixed-angle centrifuge •vacuum manifold (e.g., Vac-Man®Laboratory Vacuum Manifold,20-sample [Promega Cat.# A7231])•optional:Eluator™ Vacuum Elution Device (Cat.# A1071)Comparison of Inches of Hg to Other Pressure Measurements.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TM280 Revised 11/09Page 3Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TM280Printed in USA.Page 4Revised 11/093.Equipment, Supplies and Preparation of Solutions (continued)Before lysing cells and purifying DNA , Endotoxin Removal Wash andColumn Wash must be prepared as described below (cap tightly afteradditions):Endotoxin Removal Wash Preparation•2 preps system (Cat.# A2391): Add 5.5ml of isopropanol to the Endotoxin Removal Wash before use.•10 preps system (Cat.# A2392):Add 24ml of isopropanol to the Endotoxin Removal Wash before use.•25 preps system (Cat.# A2393):Add 57ml of isopropanol to the Endotoxin Removal Wash before use.Column Wash Preparation•2 preps system (Cat.# A2391): Add 30ml of 95% ethanol to the Column Wash before use.•10 preps system (Cat.# A2392):Add 137ml of 95% ethanol to the Column Wash before use.•25 preps system (Cat.# A2393):Add 350ml of 95% ethanol to the Column Wash before use.4.PureYield™ Plasmid Maxiprep System Protocol4.A.Preparation and Lysis of Bacterial Cell CulturesNote: Throughout the remainder of this document the supplied CellResuspension Solution (CRA), Cell Lysis Solution (CLA) andNeutralization Solution (NSB) are referred to as Cell ResuspensionSolution, Cell Lysis Solution and Neutralization Solution, respectively.Note: Perform all lysis steps at room temperature.1.Grow 100–250ml of transformed E. coli bacterial cell culture overnight(16–21 hours) at optimal culture conditions.2.Pellet the cells by centrifugation at 5,000 × g for 10 minutes at roomtemperature, and discard supernatant. Drain tubes on a paper towel toremove excess media.3.Resuspend the cell pellets in 12ml of Cell Resuspension Solution.Note:Make sure that cell resuspension is complete.4.Add 12ml of Cell Lysis Solution and mix by gently inverting the tube3–5 times. Incubate for 3 minutes at room temperature.Note:If the Cell Lysis Solution becomes too cold, SDS precipitation mayoccur, which could result in poor cell lysis. If a precipitate has formed,warm the Cell Lysis Solution to 37°C with gentle shaking before use.5.Add 12ml of Neutralization Solution to the lysed cells, cap the tube andmix by gently inverting the tube 10–15 times. It is important to completelymix the solution to ensure complete precipitation of cellular debris. Awell-mixed solution will appear flocculant, with small- to medium-sizedclumps. If the solution has not been mixed thoroughly it will instead have acoagulated appearance.6.Centrifuge the lysate at 14,000 × g for 20 minutes at room temperature in afixed-angle rotor. This centrifugation will pellet the bulk of the cellulardebris. If debris remains it can be removed using a PureYield™ ClearingColumn.Alternatively, the lysate may be centrifuged at 7,000 × g for 30 minutes.When the spin is complete, examine the cleared lysate. It should be clear,with small amounts of cell debris. If the lysate is cloudy, it should befiltered through Miracloth™ before continuing.4.B.DNA PurificationNote: Perform all purification and elution steps at room temperature.1.Assemble a column stack by placing a blue PureYield™ Clearing Columnon the top of a white PureYield™ Maxi Binding Column. Place theassembled column stack onto the vacuum manifold (Figure 1).Note:Alternatively, if the lysate is completely clear of cloudiness anddebris, the blue clearing column may be omitted, and the lysate can bepoured directly onto the binding column.Figure 1. The blue PureYield™ Clearing Column on a white PureYield™ Maxi Binding Column.The two columns sit on a vacuum manifold port.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TM280Revised 11/09Page 54594c a4.B.DNA Purification (continued)Note: Perform all purification and elution steps at room temperature.2.Pour approximately one-half of the lysate into the blue PureYield™Clearing Column (the column capacity will not allow all of the lysate to beadded at once). When processing multiple samples you will need to keeptrack of which lysate is being placed into which column to avoid cross-contamination.3.Apply maximum vacuum. The lysate will pass through the clearingmembrane in the PureYield™ Clearing Column, and the DNA will bind tothe binding membrane in the PureYield™ Maxi Binding Column. Continuethe vacuum until all the liquid has passed through both columns.4.Add the remainder of the lysate to the appropriate clearing column andallow it to pass through both columns as described above.5.Slowly release the vacuum from the filtration device before proceeding.Remove and discard the PureYield™ Clearing Column, leaving thePureYield™ Maxi Binding Column on the vacuum manifold.Wash6.Add 5ml of Endotoxin Removal Wash (as prepared in Section 3) to thePureYield™ Maxi Binding Column, turn on the vacuum and allow thevacuum to pull the solution through the column. For ease of use, thePureYield™ Maxiprep column is labeled with 5, 10, and 20ml fill levels(Figure 2), allowing you to carefully pour or pipette buffers directly intocolumns.Figure 2. PureYield™ Maxiprep column showing volume labels.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TM280Printed in USA.Page 6Revised 11/098530TANote:If the solution does not pass through the membrane, it could be dueto small amounts of particulates. If this occurs, remove the binding columncontaining the wash solution from the vacuum manifold and place it in a50ml disposable plastic centrifuge tube. Centrifuge the tube in a swingingbucket rotor for 5 minutes at 2,000 × g to allow the wash to pass throughthe membrane. After centrifugation, remove the binding column and placeit back on the vacuum manifold for the next step.7.Add 20ml of Column Wash (as prepared in Section 3) to the bindingcolumn and allow the vacuum to draw the wash through.8.Dry the membrane by applying a vacuum for 5 minutes. After drying, thetops of the DNA binding membranes should appear dry and there shouldbe no detectable ethanol odor. If the binding column membrane topsappear wet or there is a detectable ethanol odor, repeat the vacuum step todry the membrane for an additional 5 minutes.If more than six samples are being processed at once, the initial drying stepshould be increased to 10 minutes to compensate for the reduced vacuum.9.Remove the PureYield™ Maxi Binding Column from the vacuum manifold.Tap the tip of the column on a paper towel to remove excess ethanol. Wipeany excess ethanol from the outside of the tube.Elute by Vacuum (alternatively, see Elute by Centrifugation, below)Note: Elution using the Eluator™ Vacuum Elution Device results in betterDNA recovery and yield than elution by centrifugation.10.Place a 1.5ml microcentrifuge tube in the base of the Eluator™ VacuumElution Device, securing the tube cap in the open position as shown(Figure 3, Panel A).11.Assemble the Eluator™ Device and insert the DNA binding column into it,making sure the column is fully seated on the collar.12.Place the Eluator™ Device assembly, Figure 3, Panel B, onto a vacuummanifold.13.Add 1ml of Nuclease-Free Water to the binding column. Apply maximumvacuum for 1 minute or until all liquid has passed through the column.14.Remove the 1.5ml tube and save for DNA quantitation and gel analysis.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TM280 Revised 11/09Page 74.B.DNA Purification (continued)Figure 3. Elution by vacuum. Panel A. A 1.5ml microcentrifuge tube is placed in the base of the Eluator™ Vacuum Elution Device (Cat.# A1071) with the tube capsecured in the open position. Panel B . The Eluator™ Vacuum Elution Deviceassembly, including the binding column, ready for use on a vacuum manifold.Elute by CentrifugationNote:Use a room temperature centrifuge with a swinging bucket rotor forthe elute by centrifugation steps. To ensure complete passage of solutionsthrough columns, do not cap the 50ml tube during centrifugation.15.To elute the DNA place the PureYield™ Maxi Binding Column into a new50ml disposable plastic centrifuge tube. Add 1.5ml of Nuclease-Free Waterto the DNA binding membrane in the binding column.Note: If greater DNA concentration is desired, elute the plasmid in 1ml ofwater. Yield will be decreased, but concentration will be increased.16.Centrifuge the PureYield™ Maxi Binding Column in a swinging bucketrotor at 2,000 × g for 5 minutes.17.Collect the eluate from the 50ml tube and transfer to a 1.5ml tube ifdesired. Note:The recovered solution volume may be less than the amountof water added to elute the DNA.Note:To increase the DNA concentration, perform an ethanol precipitation:Add one-tenth of a volume of 3M sodium acetate (pH 5.2) and 2.5 volumes95% ethanol. Mix well and place on ice for 15 minutes. Pellet the DNA at14,000 × g for 10 minutes in a microcentrifuge. Discard the supernatant. Washthe pellet with 70% ethanol, mix well and centrifuge at 14,000 × g for 2–5minutes. Discard the ethanol, dry the pellet, then resuspend the DNA in wateror TE buffer with pipetting.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TM280Printed in USA.Page 8Revised 11/097371T AA. B.Figure 4. Concentration and yield of DNA from a variety of elution volumes.Cultures (250ml) of JM109 cells containing phMGFP plasmid were grown understandard conditions. Cells were pelleted and DNA purified using the PureYield™Plasmid Maxiprep System. Elution volumes ranged from 0.6–2ml, when eluted by centrifugation. Each data point is the average of three sample results. Note that the volumes eluted from the column are typically less than the volume added due torehydration of the membrane.5.Supplemental Information5.A.Considerations Regarding the PureYield™ Plasmid Maxiprep SystemOne of the most important aspects of any plasmid preparation is effective celllysis. Lysis is usually the limiting step, determining how much cell mass canbe processed in a plasmid preparation. If resuspended cells have too great adensity, the lysis solution will be less effective in releasing plasmid into thelysate. Exceeding the cell culture volume limit suggested by the kitmanufacturer (for PureYield™ Plasmid Maxiprep System the limit is 250ml)can lead to reduced, rather than increased, yields.It is possible to improve lysis efficiency by increasing the volumes ofresuspension, lysis and neutralization solutions used. With the PureYield™Plasmid Maxiprep System the volumes listed in the protocol (12ml) for each ofthese solutions will give effective lysis for 250ml of cell culture. Doubling theresuspension, lysis and neutralization solution volumes to 24ml will giveefficient lysis for cell culture volumes >250ml and the binding columnmembrane can support this increased volume of lysate if the cells are lysedsufficiently, allowing more plasmid to be bound and eluted. For plasmids witha low copy number, BACs and cosmids, binding capacity of the membrane isnot a limiting factor; one can load very large amounts of lysate onto thebinding column to increase yield. However, for high-copy-number plasmidsthe maximum amount of culture volume is 250ml; volumes >250ml willcontain more plasmid DNA than the binding column can bind.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TM280Revised 11/09Page 95784M A 0Elution VolumeY i e l d (µg )01002003004005006007008009001,000C o n c e n t r a t i o n (µg /m l )5.A.Considerations Regarding the PureYield™ Plasmid Maxiprep System(continued)Optimal resuspension, lysis and neutralization solution volumes will depend on the culture media and cell type used and should be determined by theindividual researcher. The PureYield™ Plasmid Maxiprep System providesenough of these solutions for 12ml of each per preparation, which is sufficient for up to 250ml cell culture. The solutions are also available for saleindividually (see Section 2 for details) for researchers interested in enhancing the effectiveness and flexibility of their plasmid preparations.The high-speed centrifugation step (Section 4.A, Step 6) is designed to remove most or all of the cellular debris from the lysate. Some debris may not pelleteffectively; in this case, it can be removed using the blue PureYield™ Clearing Column. Excessive mixing after the addition of the Cell Lysis Solution and Cell Neutralization Solution can cause the centrifuged lysate to be cloudy and this cloudiness can cause clogging of the clearing or binding columns. Cloudylysate should be recentrifuged or filtered through Miracloth™ before loading onto the column.5.B.Selection and Preparation of Plasmids and E. coli StrainsPlasmid DNA can be purified from overnight cultures of E. coli with thePureYield™ Plasmid Maxiprep System. The yield of plasmid DNA will vary depending on a number of factors, including the plasmid copy number, celldensity of bacterial culture, type of culture medium and the bacterial strainused. Plasmid copy number is an important factor affecting plasmid DNAyield. Copy number is determined primarily by the region of DNAsurrounding and including the origin of replication. This region, known as the replicon, controls replication of plasmid DNA by bacterial enzyme complexes.Some DNA sequences, when inserted into a particular plasmid, can lower the copy number of the plasmid by interfering with replication.Choose a single, well-isolated colony from a fresh Luria-Bertani (LB) agar plate (containing antibiotic) and use the colony to inoculate 1–10ml of LB media(also containing antibiotic). The inoculated medium should be incubated for8 hours at 37°C to achieve logarithmic growth. This starter culture should thenbe diluted 1:500 to inoculate a larger volume of culture media containingantibiotic, which is incubated for 12–16 hours at 37°C. An O.D.600of 2–4 forhigh-copy-number plasmids ensures that bacteria have reached the propergrowth density for harvesting and plasmid DNA isolation.5.C.Choosing a Bacterial StrainEndonuclease I is a 12kDa periplasmic protein that degrades double-stranded DNA. This protein is encoded by the gene endA. The E. coli genotype endA1refers to a mutation in the wildtype endA gene, which produces an inactiveform of the nuclease. E. coli strains with this mutation are referred to as EndA–.Table 1 contains a list of EndA–and EndA+E. coli strains.Table 1. EndA – and EndA + Strains of E. coli .Note: Using the PureYield™ Plasmid Maxiprep System, high-quality DNA iseasily obtained from both EndA+and EndA–bacterial strains.5.D.Composition of Buffers and SolutionsPromega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·Cell Resuspension Solution50mMTris-HCl (pH 7.5)10mMEDTA (pH 8.0)100μg/mlRNase A Cell Lysis Solution 0.2MNaOH 1%SDS Neutralization Solution 4.09Mguanidine hydrochloride (pH 4.2)759mMpotassium acetate 2.12M glacial acetic acid Column Wash 162.8mM potassium acetate 22.6mM Tris-HCl (pH 7.5)0.109mM EDTA (pH 8.0)Before use add 95% ethanol as directed in Section 3. Final concentrations will be approximately 60% ethanol, 60mM potassium acetate, 8.3mM Tris-HCl and 0.04mM EDTA.6.TroubleshootingFor questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information available at: . E-mail: techserv@ Symptoms Causes and CommentsPoor cell lysis Too many bacterial cells in culture medium.Use LB medium to grow bacteria. The use ofrich medium or excessive culture volumesmay lead to a biomass value too high forcomplete lysis. All media should containantibiotics at the appropriate concentration.Poor resuspension of bacterial cell pellet. Thecell pellet must be thoroughly resuspendedprior to cell lysis. Pipet or disperse (using anapplicator stick) the pellet with CellResuspension Solution. No cell clumps shouldbe visible after resuspension.No plasmid DNA Ethanol was not added to Column Wash. Preparethe Column Wash as instructed (Section 3)before beginning the procedure.Isopropanol was not added to EndotoxinRemoval Wash. Prepare the Endotoxin RemovalWash as instructed (Section 3) beforebeginning the procedure.PureYield™ Clearing Column clogged due to:•Too much cell mass. Use less biomass or non-rich medium.•Lysate not centrifuged at high enough × g.Use 14,000 × g for 20 minutesor 7,000 × g for 30 minutes.•If lysate is cloudy after centrifugation, filterthrough Miracloth™.PureYield™ Binding Column clogged due to:•Particulates in the lysate. Transfer bindingcolumn to 50ml centrifuge tube andcentrifuge in a swinging bucket rotor at roomtemperature for 5 minutes at 2,000 × g.•Insufficient vacuum. Ensure that vacuumsource is at a minimum of 650mm Hg. Lysate is cloudy SDS precipitation may have occurred:•Centrifuge at room temperature to clearlysate.•If debris not sufficiently removed bycentrifugation, filter lysate throughMiracloth™ before adding lysate to column.Symptoms Causes and CommentsDenaturation of plasmid DNA Overincubation during lysis step, Section 4.A.Total incubation of cell suspension with CellLysis Solution should not exceed 5 minutes. Genomic DNA contamination Vortexing or overmixing after addition of theCell Lysis Solution. Do not vortex samples afteraddition of Cell Lysis Solution to preventshearing of genomic DNA.Low plasmid DNA yields Overgrowth of bacterial culture bynontransformed bacteria:•Make certain that antibiotics were used in allmedia, liquid and solid. Do not culturebacteria longer than 24 hours. Optimalculture length is 12–16 hours.•Bacterial culture is too old. Inoculateantibiotic-containing media with freshlyisolated bacterial colony from an overnightplate.Some bacterial cells are more resistant to lysisthan others and may require incubation for upto 5 minutes for efficient lysis. The lysate maynot clear completely, but do not extend lysisbeyond 5 minutes as lysis for >5 minutes mayresult in nicked or single-stranded DNA.Wrong reagents used. Make certain thatColumn Wash is diluted with ethanoland the Endotoxin Removal Wash is dilutedwith isopropanol before use (Section 3). Useonly the reagents supplied with the PureYield™Plasmid Maxiprep System.Plasmid DNA yield not accurately quantitated.Quantitation by absorbance at A260mayoverestimate yield due to absorbance by avariety of contaminants such as RNA andprotein. This is especially true for low-copy-number plasmids when the ratio ofcontaminants to plasmid DNA is high. Useagarose gel/ethidium bromide quantitation.For plasmids larger than 10kb, yield may beincreased by heating the water to 65°C at theelution step. Add water to the binding columnand let sit for 1 minute. Elute as normal.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6.Troubleshooting (continued)Symptoms Causes and CommentsEthanol carryover Ethanol carryover is detected in the finalproduct. Dry the column for an additional5 minutes on the vacuum manifold.Column Wash could be present on theoutside of the column due to splashing duringthe wash step. Remove any residual ethanolfrom the outside of the column prior to elution,as directed in Section 4.B, Step 9.ing the Vac-Man®Laboratory Vacuum Manifold7.A.DescriptionThe Vac-Man®Laboratory Vacuum Manifold (Figure 5), when used in conjunction with any of the Wizard®, PureYield™ or SV Isolation Systems, is the ideal system for rapid, effective nucleic acid purification. Reliable, sturdy and easy to use, the Vac-Man®Laboratory Vacuum Manifold will process from 1 to 20 samplessimultaneously. Because each manifold comes complete with a set of 20individually controlled One-Way Luer-Lok®Stopcocks, as many or as few samples as desired can be processed at one time. The One-Way Luer-Lok®Stopcocks aredesigned to accommodate Promega Minicolumns and Maxi/Megacolumns, as well as the PureYield™ Clearing Columns and PureYield™ Maxi Binding Columns. 7.B.Setup and OperationThe Vac-Man®Laboratory Vacuum Manifold is designed for use with a smallbenchtop laboratory vacuum source (pump). The manifold is not designed for use under a high-pressure vacuum (see label for further information).Note:If you have any questions about the suitability of your vacuum source, please contact your local Promega branch office or distributor. Contact informationavailable at: or e-mail: techserv@Do not substitute chemicals. Always follow safe laboratory practices, including the use of safety glasses and a laboratory coat.1.Inspect the manifold connectors, One-Way Luer-Lok®Stopcocks and Spinlock IIAdapters, included with the manifold, for any evidence of wear or mishandling.Do not use the manifold if significant damage, such as a crack or an abrasion, isapparent.。

质粒抽提

8. 最高速再离心1分钟，除去残留液体并使痕量乙醇完全挥发。注意：倒弃收集管内液体后再离心，才能彻底去除微量的溶液IV。微量的溶液IV会影响质粒的质量。
9. 将质粒纯化柱置于1.5毫升离心管上，加入120微升溶液V至管内柱面上，放置1分钟。溶液 V 需要直接加至管内柱面中央，使液体被纯化柱吸收。如果不慎将溶液 V 沾在管壁上，一定要震动管子，使液体滑落到管底，以被纯化柱吸收。也可以用重蒸水或 MiliQ 级纯水替代溶液 V，但是水的 pH 应不小于 6.5。溶液 V 加入后放置时间稍长，对于增加质粒产量会略有帮助。
用1.5毫升离心管作为收集管。溶液V需要直接加至管内柱面中央，使液体被纯化柱吸收。如果不慎将溶液V沾在管壁上，一定要震动管子，使液体滑落到管底，以便被纯化柱吸收。也可以用重蒸水或MiliQ级纯水替代溶液V，但是水的pH应不小于6.5。溶液V加入后放置时间稍长，对于增加质粒产量会略有帮助。如想得到较高浓度的质粒，可以加入80微升溶液V 洗脱。 15. 最高速离心1分钟，所得液体即为转细胞级超纯质粒。通常所得质粒浓度为0.1-0.3mg/ml左右。如果想得到高浓度的质粒，可以采用常规的乙醇沉淀方法浓缩质粒。
3. 每管加入250微升溶液II，轻轻颠倒离心管4-6次，使细菌完全裂解，溶液透明。切勿vortex！vortex或其它剧烈操作会导致基因组DNA断裂，易导致最终所得质粒被基因组DNA污染。颠倒4-6次后，溶液应变得透明，无团块或絮状物。如果加入溶液I后细菌没有完全散开，那么颠倒4-6次后，可能还会有团块或絮状物。遇到有少量团块或絮状物产生的情况，可以增加颠倒次数3-5次，再室温放置2-3分钟，但总裂解时间不可超过5分钟。
10. 最高速离心1分钟，所得液体中加入600微升溶液VI，混匀后加到原质粒纯化柱内。加入溶液VI后必须混匀！可vortex或颠倒混匀，混匀后切勿离心。

无内毒素质粒 DNA 大量提取试剂盒

5 12,000 rpm 室温离心 12min，收集上清。 6 将上清置于 DNA 纯化柱中，静置 5 min。
●
如果收集的上清液过多，超过 DNA 纯化柱容积（15 ml），可将上清分次加入 DNA 纯化柱中。
7 12,000 rpm 离心 2min，弃滤液。
●
此时质粒 DNA 被吸附于 DNA 纯化柱中的硅胶膜上。
溶液 W 初次使用前用无水乙醇按 1: 1.5 稀释，即含 60% 乙醇。 ● 溶液Ⅱ、溶液Ⅲ、溶液 PB 中含碱和变性剂，请不要直接接触皮肤。 ● 上述产品组分均可单独购买，详见产品索引。
质量控制从大肠杆菌提取 pGEM 质粒 DNA。提取的质粒DNA 质量通过琼脂糖凝胶电泳、限制性酶切和序列测定分析。保存条件 RNase A：可室温保存 1 年以上。RNase A 为浑浊溶液。初次使用本试剂盒时，请将 RNase A 全部加入到溶液Ⅰ中，均匀混合后于 4℃保存。可保存 6 个月。其他试剂：室温保存。若溶液Ⅱ出现沉淀，请于 37℃保温溶解。待恢复至室温后使用。沉淀的出现不会影响质粒 DNA 的纯化结果。注意事项-------------------------------------------------------------------------------------------------------------------------------------------------------------● ● ● ●
● ● ●
若溶液Ⅱ出现沉淀，请于 37℃保温溶解。待恢复至室温后使用。沉淀的出现不可剧烈混和，否则会使染色体 DNA 断裂。此步骤不宜超过 5 min。
不会影响质粒 DNA 的纯化结果。

质粒大提试剂盒说明书

北京索莱宝科技有限公司质粒大提试剂盒说明书货号：D1110规格：10次保存：常温保存，复检期一年。

试剂盒内容：RNaseA(10mg/ml)1ml溶液Ⅰ60ml溶液Ⅱ60ml溶液Ⅲ80ml漂洗液2×15ml洗脱液30ml吸附柱10个收集管(50ml)20个说明书1份产品说明：本试剂盒采用碱裂解法裂解细胞，根据离心吸附柱在高盐状态下特异性地结合溶液中的DNA的原理特异性提取质粒DNA。

离心吸附柱中采用的硅基质材料能高效、专一地吸附DNA，可最大限度去除杂质蛋白及细胞中其他有机化合物，一般从50-100ml大肠杆菌LB培养液中，可快速提取200-300μg高纯度高拷贝的质粒DNA，提取率达85-90%。

使用本试剂盒提取的质粒DNA可适用于各种常规操作，包括酶切、PCR、测序、连接和转化等试验。

使用前请先在漂洗液中加入无水乙醇，加入体积请参照瓶体上的标签。

溶液Ⅰ在使用前先加入RNaseA（将试剂盒中提供的RNaseA全部加入），混匀，置于2-8℃保存。

如非指明，所有离心步骤均为使用台式离心机在室温下离心。

操作步骤:第1页共3页1.收集50-100ml过夜培养的菌液11000rpm离心10分钟，尽量吸除上清(菌液较多时可以通过多次离心将菌体沉淀收集到一个离心管中)。

2.向留有菌体沉淀的离心管中加入5ml溶液Ⅰ(请先检查是否已加入RNaseA)，使用移液器或涡旋振荡器彻底悬浮细菌细胞沉淀。

注意：如果菌块未彻底混匀，会影响裂解导致质粒提取量和纯度偏低。

3.向离心管中加入5ml溶液Ⅱ，温和地上下翻转6-8次使菌体充分裂解。

注意：①翻转一定要温和，以免污染细菌基因组DNA。

②作用时间不要超过5分钟，以免质粒受到破坏。

4.向离心管中加入7ml溶液Ⅲ，立即温和地上下翻转6-8次，充分混匀，此时会出现白色絮状沉淀(如菌液过多，可在此步放置5分钟，以尽可能的降解RNA)。

11000rpm离心10分钟，用移液器小心地将上清转移到另一个干净的离心管中，注意尽量不要吸出沉淀。

超速无内毒素质粒大量高效提取试剂盒

产品说明书◆超速无内毒素质粒大量提取试剂盒◆目录号1217◆使用手册◆实验室使用，仅用于体外超速无内毒素质粒大量提取试剂盒试剂盒组成、储存、稳定性：试剂盒组成保存10次（121701）20次（121702）溶液P1 室温50ml 100ml溶液P2 室温50 ml 100 ml溶液N3 室温50 ml 100 ml溶液WB 室温15 ml 30 ml沉淀液A 室温18 ml 35 ml沉淀液B 室温 1.1 ml 2.5 ml内毒素清除剂-20℃ 1.5ml 1.5 ml本试剂盒在室温储存18个月不影响使用效果。

储存事项：1.环境温度低时溶液P2中SDS可能会析出出现浑浊或者沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。

2.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。

产品介绍：本试剂盒在改良碱裂解法基础上，采用本公司独家研发的溶液配方和内毒素清除试剂，只需要几次简单离心去除蛋白质、多糖、内毒素、RNA等杂质，获得高质量的质粒DNA。

纯化DNA的OD260/280通常在1.8左右，得到的质粒可直接用于酶切、转化、PCR、体外转录、测序等各种分子生物学实验。

同时内毒素含量极低，可直接应用于细胞转染甚至动物体内实验等对DNA纯度要求很高的工作中。

纯化后期过程均在Eppendorf管中操作，方法简单，不需特殊设备，无需过柱，不用酚氯仿抽提。

本产品是目前市面上最简捷快速的无内毒素质粒大提产品，属于公司独家产品。

- 1 -操作步骤：1.取过夜培养菌<140 ml菌液，装入合适的离心瓶中，12,000 x g离心3 min沉淀菌体，完全弃除上清。

2.加入5 ml 溶液P1，充分混悬震荡菌体沉淀，使其完全分散开，至无絮块存在。

细菌悬液移入50 ml离心管中。

如果有未彻底混匀的菌块，会影响裂解，导致提取量和纯度偏低。

3.加入5 ml 溶液P2，轻轻颠倒离心管6~8次，室温放置5 min，使细菌完全裂解，溶液透明。

无内毒素质粒大量快速提取试剂盒操作方法及步骤说明书

杭州昊鑫生物科技股份有限公司 htpp://EndoFree Plasmid Maxi Kit无内毒素质粒大量快速提取试剂盒目录号：PL13试剂盒组成、储存、稳定性：试剂盒组成保存10次(PL1301)RNaseA（10mg/ml）-20℃750µl溶液P1 4℃77 ml溶液P2 室温77 ml溶液N3 室温77 ml内毒素清除剂-20℃25 ml漂洗液WB 室温25 ml X 2第一次使用前按说明加指定量乙醇洗脱缓冲液EB 室温20 ml吸附柱DC 室温10个收集管（50ml）室温10个本试剂盒在室温储存12个月不影响使用效果。

内毒素清除剂常温运输，4度可以保存一个月，长期保存放-20℃。

储存事项:1.第一次使用时，将试剂盒所带全部的RNase A加入溶液P1后（终浓度100ug/ml）置于4℃保存。

如果溶液P1中RNase A失活，提取的质粒可能会混杂有微量RNA 残留，在溶液P1中补加RNase A即可。

2.环境温度低时溶液P2中SDS可能会析出出现浑浊或者沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。

3.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。

产品介绍：本试剂盒采用改进SDS-碱裂解法裂解细胞，粗提物通过独特的内毒素清除剂选择性结合离心除去内毒素，然后离心吸附柱内的硅基质膜在高盐、低pH值状态下选择性地结合溶液中的质粒DNA，再通过漂洗液将杂质和其它细菌成分去除，最后低盐、高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。

产品特点：1.离心吸附柱内硅基质膜全部采用进口世界著名公司特制吸附膜，柱与柱之间吸附量差异极小，可重复性好。

克服了国产试剂盒膜质量不稳定的弊端。

2.不需要使用有毒的苯酚，氯仿等试剂，也不需要乙醇沉淀。

快速，方便，从150-300ml大肠杆菌LB((Luria-Bertani)培养液中，可快速提取0.2-1.5mg纯净的高拷贝质粒DNA，提取率达80-90 %。

无内毒素质粒小提中量试剂盒操作方法及步骤说明书

杭州昊鑫生物科技股份有限公司 htpp://EndoFree Plasmid Midi Kit无内毒素质粒小提中量快速提取试剂盒目录号：PL10试剂盒组成、储存、稳定性：试剂盒组成保存50次(PL1001)平衡液室温5mlRNaseA（10mg/ml）-20℃250µl溶液P1 4℃25 ml溶液P2 室温25 ml溶液N3 室温25 ml内毒素清除剂-20℃10 ml漂洗液WB 室温15 ml第一次使用前按说明加指定量乙醇洗脱缓冲液EB 室温15ml吸附柱AC 室温50个收集管（2ml）室温50个本试剂盒在室温储存12个月不影响使用效果。

内毒素清除剂常温运输，4度可以保存一个月，长期保存放-20℃。

储存事项:1.第一次使用时，将试剂盒所带的全部RNase A加入溶液P1后（终浓度100ug/ml）置于2-8℃保存。

如果溶液P1中RNase A失活，提取的质粒可能会有微量RNA 残留，在溶液P1中补加RNase A即可。

2.环境温度低时溶液P2中SDS可能会析出浑浊或者沉淀，可在37℃水浴加热几分钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。

3.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。

产品介绍：本试剂盒采用改进SDS-碱裂解法裂解细胞，通过独特的内毒素清除剂选择性结合离心除去内毒素，然后离心吸附柱内的硅基质膜在高盐、低pH值状态下选择性地结合溶液中的质粒DNA，再通过去蛋白液和漂洗液将杂质和其它细菌成分去除，最后低盐、高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。

产品特点：1.离心吸附柱内硅基质膜全部采用进口世界著名公司特制吸附膜，柱与柱之间吸附量差异极小，可重复性好。

克服了国产试剂盒膜质量不稳定的弊端。

2.独特工艺配方清除内毒素，内毒素含量极低（<0.1 EU/μg DNA），细胞转染效果极佳。

也可直接用于酶切、转化、PCR、体外转录、测序、等各种分子生物学实验。

无内毒素质粒小量提取试剂盒使用说明

9、向吸附柱中加入 700ul 漂洗液 (使用前请先检查是否已加入无水乙醇 )， 12000rpm 离心 1min，弃废液，将吸附柱放入收集管中。
10、向吸附柱中加入 500ul 漂洗液， 12000rpm 离心 1min，弃废液，将吸附柱放入收集管中。
3、向离心管中加入 200ul 溶液 P2，温和地上下翻转 6-8 次使菌体充分裂解。注意：混匀一定要温和，以免污染细菌基因组 DNA，此时菌液应变得清亮粘稠，作用时间不要超过 5 min，以免质粒受到破坏。
2. 洗脱缓冲液体积不应少于 50ul，体积过小影响回收效率；洗脱液的

pH 值对洗脱效率也有影晌，若需要用水做洗脱液应保证其 pH 值在 8.0 左右 (可用 NaOH 将水的 pH 值调至此范围 )， pH 值低于 7. 0 会降低洗脱效率。
由于质粒dna本身的性质清除过程可导致部分质粒dna丢失但内毒素却能得到最大限度清所有溶液应用无内毒素的高纯水配制所有器械材料均应不含内毒素玻璃器皿可高温烘烤非挥发性水溶液可高压处理

无内毒素质粒小量提取试剂盒使用说明货号： D1140 规格： 50T/100T 保存：常温干燥保存，复检期为一年。试剂盒内容：

有机化合物。内毒素清除剂可最大限度地除去内毒素。从 1-5ml 大肠杆菌 LB 培养液中，可快速提取 5-15μ g 高纯度质粒 DNA，提取率达 85-90%。使用本试剂盒提取的质粒 DNA 纯度高，可直接用于细胞转染等要求较高的实验，以及其他各种常规操作，包括酶切、 PCR、测序、连接和转化等试验。

质粒DNA大提试剂盒-经典法

技术咨询电话：400-607-9999质粒DNA大提试剂盒-经典法Classic Max Plasmid DNAout货号: M090110产品及特点本试剂盒是用于质粒DNA大量制备与纯化的试剂盒。

菌体先经碱裂解法处理，再通过离心吸附柱，专一结合DNA，最后洗去杂质，高效快速提取质粒DNA，全套操作可以在30分钟之内完成。

使用本试剂盒可从50-100 mL过夜培养的菌液纯化得到高达300-500 ug的高纯度质粒DNA（OD260/OD280 = 1.8-2.0），可以直接用于酶切、转化、测序及PCR等。

1.快速、步骤少，整个操作在半个小时之内完成。

2.纯度高，采用本试剂盒提取的质粒OD比值在1.8-2.0之间。

3.产量高，每毫升过夜培养的细菌可以提取到2-5 ug/mL。

4.用途广，适用于低拷贝和高拷贝质粒。

5.价格低，比多数国内同类产品的价格更便宜。

规格及成分5次包装成份溶液A 30mL溶液B 30mL溶液C 40mLRNase A溶液1.5 mL（2 mg/mL）通用洗柱液100mL大提离心吸附柱5套通用洗脱液10mL使用手册1份运输及保存RNase A溶液需要4℃保存，其余成分可室温保存，如有沉淀可加热溶解后再使用。

技术咨询电话：400-607-9999使用方法1.先将全部RNase A溶液全部加入到溶液A中，摇匀后再取用，未用完的溶液A最好在4℃保存。

2.用250 mL离心管收集100-300 mL菌液，5,000 rpm离心10分钟，去除上清。

3.往细菌沉淀中加入6 mL溶液A（含RNase A），充分振荡悬浮。

注意：充分重悬细胞沉淀（无块状物）对于获得高的质粒产量十分重要。

4.加入6 mL 溶液B，温和翻转10 余次至透明。

若混合液不透明，应减少细菌的用量或室温放置5-10分钟直到裂解液变透明。

注意：避免剧烈振荡，否则细菌基因组DNA将断裂为小片段，与质粒难以分离，污染质粒DNA。

中提试剂盒说明书

实验操作步骤1.将过夜培养内含高拷贝质粒的细菌25-50ml，或内含低拷贝质粒50-100ml细菌, 5000转/分, 离心10分钟，彻底去除上清。

2.加入1.5 ml Solution I，用枪头或振荡器充分悬浮细菌。

3.加入3 ml Solution II，立即上下颠倒5－10次，使细菌裂解，室温放置至溶液呈透明状。

注意：不能用振荡器混，也不能用力混，否则会出现Genomic DNA污染。

4.加入6 ml Solution III，立即上下颠倒5－10次，使之充分中和, 室温放置5分钟。

注意：步骤2和3在冰上操作效果更佳。

注意：不能用振荡器混，也不能用力混，否则会出现Genomic DNA污染。

5.12,000 x g高速离心10分钟（速度高些更好，取决于离心机和离心管的类型）。

6.将3SM柱放入50ml收集管，将步骤5中的部分上清转移到3SM柱中；如离心后溶液表面没有漂浮物，上清可以直接倒入3SM柱。

室温放置5分钟；12000x g离心2分钟。

注意：转移上清时不要吸取沉淀，否则会出现Genomic DNA和蛋白质污染。

7.取出3SM柱，去掉收集管中的废液，将3SM柱放入同一支收集管中，吸取5 ml Wash SolutionI 到3SM柱，12000x g离心2分钟。

8.取出3SM柱，去掉收集管中的废液，将3SM柱放入同一支收集管中，吸取5 ml Wash Solution II 到3SM柱，12000x g离心2分钟。

9.取出3SM柱，去掉收集管中的废液，将3SM柱放入同一支收集管中，吸取5 ml Wash Solution 到3SM柱，12000x g离心2分钟。

(Wash Solution中需要加入50ml乙醇)。

10.重复步骤9一次。

11.取出3SM柱，去掉收集管中的废液，将3SM柱放入同一支收集管中，12000转/分室温离心2分钟。

12.将3SM柱放入干净的50ml的离心管中，加1000-1500ml TE室温下放置2分钟，12000x g 离心2分钟。

天根RNA提取说明书

2) 胰蛋白酶处理法：确定细胞数量，吸除培养基，用PBS 洗涤细胞，吸除PBS ，向细胞中加入含有0.1-0.25%胰蛋白酶的PBS 处理细胞，当细胞脱离容器壁时，加入含有血清的培养基失活胰蛋白酶，将细胞溶液转移至RNase-Free 的离心管中，300×g 离心5 min ，收集细胞沉淀，仔细吸除所有上清。

注意：收集细胞时一定要将细胞培养液去除干净，否则会导致裂解不完全，造成RNA 的产量降低。

d. 细胞悬液：离心取细胞。

每5×106-107动物细胞和植物细胞加入1 ml TRNzol-A +。

加入TRNzol-A +前不要洗涤细胞，以免降解mRNA 。

e. 血液处理：直接取新鲜的血液，加入3倍体积TRNzol-A +(推荐0.25ml 全血加入0.75 mlTRNzol-A +)，充分振荡混匀。

2. 将匀浆样品在15-30℃放置5 min ，使得核酸蛋白复合物完全分离。

3. 可选步骤：4℃ 12,000 rpm(~13,400×g) 离心10 min ，取上清。

注意：如果样品中含有较多蛋白、脂肪、多糖或肌肉、植物结节部分等，可离心去除。

离心得到的沉淀中包括细胞外膜、多糖、高分子量DNA ，上清中含有RNA 。

处理脂肪组织样品时，上层是大量油脂，应除去。

取澄清的匀浆溶液进行下一步操作。

4. 每使用1 ml TRNzol-A +加0.2 ml 氯仿，盖好管盖，剧烈振荡15 sec ，室温放置3 min 。

注意：如不能旋涡混匀，可手动快速颠倒混匀2 min 。

5. 4℃ 12,000 rpm(~13,400×g)离心10-15 min 。

样品会分成三层：黄色的有机相，中间层和上层无色的水相，RNA 主要在水相中，把水相（约500 μl ）转移到新管中。

(如果要分离DNA 和蛋白质，可向天根公司索取提取方法)。

6. 在得到的水相溶液中加入等体积异丙醇，混匀，室温放置20-30 min 。

去内毒素质粒小提试剂盒说明书

去内毒素质粒小提试剂盒说明书试剂盒组成50次100次RNase A 100ul 200ul内毒素清除剂20ml 40ml溶液P1 10ml 20ml溶液P2 10ml 20ml溶液P3 10ml 20ml结合液30ml 60ml漂洗液15ml 15ml×2洗脱液10ml 20ml吸附柱50个100个收集管50个100个说明书1份1份注：该试剂盒置于室温（15℃-25℃）干燥条件下可保存12个月，更长时间的保存可置于2℃-8℃。

产品简介：本试剂盒采用碱裂解法裂解细胞，通过离心吸附柱在高盐状态下特异性地结合溶液中的DNA的特性提取质粒DNA。

离心吸附柱中采用的硅基质材料能高效、专一地吸附DNA，可最大限度去除杂质蛋白及细胞中其他有机化合物，同时加入Solarbio公司特制的内毒素清除剂，可最大限度地去除内毒素。

从1-5ml大肠杆菌LB培养液中，可快速提取5-15μg纯净地高拷贝质粒DNA，提取率达85-90％。

使用本试剂盒提取的质粒DNA纯度高，可直接用于细胞转染等要求较高的试验，以及其它一些常规试验如酶切、PCR、测序、连接和转化等试验。

本试剂盒无需使用酚、氯仿等有毒试剂，操作安全。

操作步骤：使用前请先在漂洗液中加入无水乙醇，加入体积请参照瓶上的标签。

溶液P1在使用前先加入RNaseA（将试剂盒中提供的RNaseA全部加入），混匀，置于2-8℃保存。

如非指明，所有离心步骤均为使用台式离心机在室温下离心。

1、取1-5ml细菌培养物，12000rpm离心1min，尽量吸除上清（菌液较多时可以通过多次离心将菌体沉淀收集到一个离心管中）。

2、向留有菌体沉淀的离心管中加入200μl溶液P1(请先检查是否已加入RNaseA），使用移液器或涡旋振荡器彻底悬浮细菌细胞沉淀。

注意：如果菌块未彻底混匀，会影响裂解导致质粒提取量和纯度偏低。

3、向离心管中加入200ul溶液P2，温和地上下翻转6-8次使菌体充分裂解。

去内毒素质粒小提试剂盒说明书

产品简介：本试剂盒采用碱裂解法裂解细胞，通过离心吸附柱在高盐状态下特异性地结合溶液中的DNA的特性提取质粒DNA。

从1-5ml大肠杆菌LB培养液中，可快速提取5-15μg纯净地高拷贝质粒DNA，提取率达85-90％。

使用本试剂盒提取的质粒DNA纯度高，可直接用于细胞转染等要求较高的试验，以及其它一些常规试验如酶切、PCR、测序、连接和转化等试验。

本试剂盒无需使用酚、氯仿等有毒试剂，操作安全。

操作步骤：使用前请先在漂洗液中加入无水乙醇，加入体积请参照瓶上的标签。

溶液P1在使用前先加入RNaseA（将试剂盒中提供的RNaseA全部加入），混匀，置于2-8℃保存。

如非指明，所有离心步骤均为使用台式离心机在室温下离心。

1、取1-5ml细菌培养物，12000rpm离心1min，尽量吸除上清（菌液较多时可以通过多次离心将菌体沉淀收集到一个离心管中）。

2、向留有菌体沉淀的离心管中加入200μl溶液P1(请先检查是否已加入RNaseA），使用移液器或涡旋振荡器彻底悬浮细菌细胞沉淀。

注意：如果菌块未彻底混匀，会影响裂解导致质粒提取量和纯度偏低。

3、向离心管中加入200ul溶液P2，温和地上下翻转6-8次使菌体充分裂解。

天根DP117-无内毒素质粒大提试剂盒说明书

For personal use only in study and research; not for commercial use产品简介本试剂盒采用独特的硅胶模吸附技术，高效专一地结合质粒DNA。

同时采用特殊的溶液P4和过滤器CS1，可有效的出去内毒素、蛋白质杂志；整个提取过程仅需1h，方便快捷。

使用本试剂盒提取的质粒DNA可适用于各种常规操作，包括酶切、PCR、测序、连接、转化和细胞转染多种细胞等试验。

推荐每次菌液使用量：高拷贝质粒推荐使用量为100ml，得率一般在500-1500μg左右；低拷贝质粒推荐使用量为200ml，得率一般在200-600μg左右。

注意事项：请务必在使用本试剂盒之前阅读此注意事项。

1.溶液P1在使用前先加入RNase A （将试剂盒中提供的RNase A全部加入)，混匀，置于2-8℃保存。

2.第一次使用前应按照试剂瓶标签的说明先在漂洗液PW中加入无水乙醇。

3.使用前先检查平衡液BL，溶液P2和P4是否出现结晶或者沉淀，如有结晶或者沉淀现象，可在37℃水浴中加热几分钟，即可恢复澄清。

4.注意不要直接接触溶液P2和P4，使用后应立即盖紧盖子。

5.使用过滤器时请将推柄小心缓慢地从过滤管中抽出，避免滤膜因压力而松动。

6.提取的质粒量与细菌培养浓度，质粒拷贝数等因素有关。

如果所提质粒为低拷贝质粒或大于10kb的大质粒，应加大菌体使用量，同时按比例增加P1、P2、P4的用量；洗脱缓冲液推荐在65-70℃水浴中预热，可以适当延长吸附和洗脱时间，以提高提取效率。

7.实验前使用平衡液BL处理吸附柱，可以最大限度激活硅基质膜，提高得率。

8.用平衡液处理过的柱子最好立即使用，放置时间过长会影响使用效果。

质粒DNA 浓度及纯度检测得到的质粒DNA可用琼脂糖凝胶电泳和紫外分光光度计检测浓度与纯度。

OD260值为1相当于大约50μg/ml双链DNA。

纯化的质粒DNA OD260/OD280通常在1.8-2.0左右，可直接应用于细胞转染甚至动物体内实验等对DNA纯度要求很高的实验中。