DNA的溶解度问题.doc

乙醇沉淀法是一种常用的技术集中的盐在水溶液中的核酸（DNA或RNA）准备。 The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution.其基本做法是，加入盐和乙醇水溶液，这迫使核酸沉淀出的解决方案。 The precipitated nucleic acid can then be separated from the rest of the solution by centrifugation.然后可以休息的解决方案，通过离心分离沉淀核酸。 The pellet is washed in cold 70% ethanol then after a further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being resuspended in clean aqueous buffer.在寒冷的70乙醇洗涤沉淀，然后再经过离心步骤乙醇被删除，并允许核酸颗粒干燥，然后在干净的缓冲溶液中悬浮。 So how does this work?那么如何工作的呢？ A bit about solubility 关于溶解度的位. First we need to know why nucleic acids are soluble in water.首先，我们需要知道为什么核酸是易溶于水。 Water is a polar molecule it has a partial negative charge near the oxygen atom due the unshared pairs of electrons, and partial positive charges near the hydrogen atoms (see the diagram on the right).水是极性分子 - 它有一个部分负电荷的氧原子附近，由于共用电子对，部分正电荷的氢原子（见右图）附近。 Because of these charges, polar molecules, like DNA or RNA, can interact electrostatically with the water molecules, allowing them to easily dissolve in water.极性分子，如DNA或RNA，因为这些费用，可以与水分子的静电相互作用，使他们能够很容易溶解于水。 Polar molecules can therefore be described as hydrophilic and non-polar molecules, which cant easily interact with water molecules, are hydrophobic.极性分子，因此可以被描述为亲水性和非极性分子，不能轻易与水分子相互作用，疏水。 Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone.核酸是亲水性由于带负电荷的磷（PO3-）组沿糖磷酸骨架。 The role of the salt 盐的作用. Ok, so back to the protocol.好吧，这样的协议。 The role of the salt in the protocol is to neutralize the charges on the sugar phosphate backbone.盐在协议中的作用是消除糖磷酸骨架上的收费。 A commonly used salt is sodium acetate.一种常用的盐是醋酸钠。 In solution, sodium acetate breaks up into Na+ and CH3COO-.在溶液中，分解成Na +和醋酸 - 醋酸钠。 The positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water.带正电的钠离子中和负电荷的核酸PO3组，分子远低于亲水性，因此多不溶于水。 The role of the ethanol 乙醇的作用. The electrostatic attraction between the Na+ ions in solution and the PO3- ions are dictated by Coulombs Law , which is affected by the dielectric constant of the solution.钠离子在溶液中和PO3-离子之间的静电吸引力， 库仑定律 ，这是解决方案的电介质常数的影响决定。 Water has a high dielectric constant, which makes it fairly difficult for the Na+ and PO3- to come together.水具有较高的介电常数，这使得它相当困难Na +和PO3-走到一起。 Ethanol on the other hand has a much lower dielectric constant, making it much easier for Na+ to interact with the PO3-, shield its charge and make the nucleic acid less hydrophilic, causing it to drop out of solution.另一方面乙醇低得多的介电常数，使得它的Na +更容易互动，PO3-，它的电荷屏蔽，使核酸的亲水性，使其下降了解决方案。 The role of temperature 温度的作用. Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (eg -20 or -80C) is commonly cited in protocols as necessary in protocols.作为必要的协议，在协议中普遍提及的核酸/盐/乙醇的混合物在低温（如-20或-80C）的孵化。 However, according to Maniatis et al (Molecular Cloning, A Laboratory Manual 2nd Edition 2nd edition? I need to get a newer version!), this is not required, as nucleic acids at concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient.然而，根据，曼尼阿蒂斯等 （分子克隆实验手册第二版.第二版- ？！我需要得到一个新的版本），这是不是必需的，因为20ng/mL低浓度核酸沉淀在0-4C，所以在冰上潜伏期为15-30分钟就足够了。 The wash step with 70% ethanol 用70乙醇洗涤步骤. This step is to wash any residual salt away from the pelleted DNA.这一步是洗任何剩余的盐颗粒的DNA。 A few tips on nucleic acid precipitation 核酸沉淀的一些技巧. Choice of salt 盐的选择 Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations使用常规的DNA沉淀醋酸钠 （0.3M终浓度，pH值5.2） Use Sodium chloride (0,2M final conc) for DNA samples containing SDS since NaCl keeps SDS soluble in 70% ethanol so it wont precipitate with the DNA.使用DNA含有SDS自氯化钠保持SDS在70乙醇中易溶，所以它不会沉淀的DNA样本， 氯化钠 （0,2米决赛浓度）。 Use Lithium Chloride (0.8M final conc) for RNA.使用氯化锂 （0.8M终浓度）的RNA。 This is because 2.5-3 volumes of ethanol should be used for RNA precipitation and LiCl is more soluble in ethanol than NaAc so will not precipitate, but beware chloride ions will inhibit protein synthesis and DNA polymerase so LiCl is no good for RNA preps for in vitro translation or reverse transcription.这是因为2.5-3乙醇卷应该被用于RNA沉淀和LiCl是比乙酸钠溶于乙醇，所以不会沉淀，但要小心 - 氯离子会抑制蛋白质的合成和DNA聚合酶，使氯化锂的RNA PREPS没有好的在体外翻译或反转录。 In these cases, use NaAc.在这种情况下，使用醋酸钠。 Use Ammonium acetate (2M final conc) for the removal of dNTPs, but do not use for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme.用于去除dNTPs浓度的醋酸铵 （2M终浓度），但不使用T4聚核苷酸激酶反应制备的DNA铵离子抑制酶。 To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides) 在低浓度或小核酸件（少于100个核苷酸）的降水以增加产量 Add MgCl2 to a final concentration of 0.01M氯化镁的终浓度为0.01M Increase the time of incubation ice before centrifugation to 1 hour.潜伏期冰离心前的时间增加至1小时。 Phenol/Chloroform Extraction酚/氯仿抽提 and Ethanol Precipitation和乙醇沉淀 DESCRIPTION描述 Phenol/Chloroform Extraction酚/氯仿抽提 One of the most commonly used and useful methods for isolation and concentration of DNA and RNA from aqueous solutions is phenol/chloroform extraction followed by ethanol precipitation.最常用的和有益的，从水溶液中的DNA和RNA的分离和浓度的方法之一是酚/氯仿抽提，乙醇沉淀。 During organic extraction, protein contaminants are denatured and partition either with the organic phase or at the interface between organic and aqueous phases, while nucleic acids remain in the aqueous phase.在有机萃取，蛋白质污染物变性和分区与有机相或有机相和水相之间的界面，而留在水相中的核酸。 Phenol used in this protocol is buffered to prevent oxidized products in the phenol from damaging the nucleic acids.在这个协议中使用的苯酚缓冲，以防止破坏核酸在苯酚氧化的产品。 Be aware that phenol can cause severe chemical burns on skin and will damage clothing.要知道，酚可引起严重的化学烧伤，皮肤上，会损伤衣物。 Wear gloves, safety glasses, and a laboratory coat when working with phenol.与苯酚工作时戴手套，安全眼镜，实验室大衣。 In the method presented here, phenol/chloroform (50%/50%; v/v) is recommended for extraction.建议在这里提出的方法，酚/氯仿（50/ 50V / V） 提取 。 In most cases, this mixture provide在大多数情况下，这种混合物提供 good protein denaturation and a tighter interphase between the aqueous and organic phases. 良好的蛋白质变性和水相和有机相之间的更紧密相间。 If there is a problem with excessive foaming during the extraction, isoamyl alcohol can be added to obtain an organic composition of phenol/chloroform (50%/49%)/isoamyl alcohol (I%). 提取过程中过多的泡沫如果有一个问题，异戊醇可以增加获得的酚/氯仿（50/ 49）/异戊醇（）的有机组成。 Conventional Ethanol Precipitation传统的乙醇沉淀 During the ethanol precipitation, salts and other solutes such as residual phenol and chloroform remain in solution while nucleic acids form a white precipitate that can,easily be collected by centrifugation.在乙醇沉淀，盐和其他溶质，如残余苯酚和氯仿溶液中保持，而核酸形成白色沉淀物，可以很容易地通过离心收集。 If the aqueous volume is less than 450 pL, the reaction can be performed in a microcentrifuge tube.如果水的体积小于450 PL，反应可在离心管中。 This is the most convenient format for performing organic extractions and ethanol precipitations.这是最方便的格式进行有机提取物和乙醇沉淀。 For larger volumes, multiple microcentrifuge tubes can be used, or the reaction can be scaled up.对于体积较大，可以使用多种离心管，可以扩展或反应。 When scaling the reaction up, use tightly capped polypropylene tubes for the phenol/chloroform extraction, and centrifuge at 2500 rpm at room temperature to resolve phases. Polystyrene tubes cannot withstand the phenol/chloroform. Ethanol precipitation can be performed in 15- or 30-mL Corex tubes, and the precipitate collected by centrifugation at 10,000 xg for 15 min at 4 OC.当缩放的反应了，使用的酚/氯仿提取，并离心机盖紧聚丙烯管在2500在室温下转速来解决阶段聚苯乙烯管能不能承受的酚/氯仿乙醇沉淀可以将在15个执行- 。或30 -毫升的Corex管，沉淀15分钟，在4离心收集10,000 XG。 It is recommended that Corex tubes be acid washed before use by immersion in 50% nitric acid for 1 hr, followed by thorough rinsing in distilled water, and autoclaving for 20 min.据建议，Corex公司管是使用在 50硝酸浸泡在蒸馏水彻底清洗，高压灭菌20分钟，1小时前洗酸。 In our hands, nucleic acid fragments and oligonucleotides longer than 15 nucleotides can be efficiently precipitated using this protocol.在我们的手，核酸片段和寡核苷酸超过15个核苷酸可以有效地沉淀，使用此协议。 For efficient precipitation, the nucleic acid concentration should be at least 10 gg/mL.为了有效降水，核酸浓度应该是至少10 GG /毫升。 Lower concentrations of nucleic acids can be precipitated, but the recovery may not be quantitative.可以沉淀核酸的浓度较低，但经济复苏可能不定量。 To precipitate lower nucleic acid concentrations, incubate the precipitate at -20 C (or on dry ice) for 4 hr to overnight, and centrifuge for 30 min to collect the precipitate.沉淀核酸浓度较低，在-20“C”（或干冰）孵育4小时至过夜，离心30分钟，收集沉淀的沉淀。 Alternatively, nanogram quantities of nucleic acid can be efficiently precipitated by adding yeast tRNA carrier to the solution to obtain a nucleic acid concentration of 10 g/mL before initiating the extraction and precipitation procedure.另外，可以有效地沉淀核酸纳克数量加入酵母tRNA载波解决方案前发起的提取和降水过程，获得了10克/毫升的核酸浓度。 The presence of TRNA carrier is typically not a problem, except when the carrier will interfere with subsequent enzymatic manipulations of the sample (eg, if a DNA fragment will be end-labeled with T4 polynucleotide kinase). tRNA的载体的存在通常是没有问题的，除承运人时，会干扰随后的酶样品的操作（例如，如果将DNA片段，用T4多核苷酸激酶末端标记）。 The recommended salt for most routine applications of this method is 0.3 M sodium acetate (final concentration), which is more soluble in ethanol than 0.3 M sodium chloride and therefore less likely to precipitate with the nucleic acid sample.这种方法最常规应用的建议盐0.3 M醋酸钠（终浓度），这是更大于0.3 m氯化钠溶于乙醇，因此不太可能与核酸样品沉淀。 For samples containing sodium dodecyl sulfate (SDS), the recommended salt is 0.2 M sodium chloride, since the SDS is soluble in ethanol under these conditions.对于含有十二烷基硫酸钠（SDS）的样品，建议的盐是氯化钠0.2 mol以来，SDS是在这些条件下易溶于乙醇。 For removal of triphosphates (labeled or otherwise), 2 M ammonium acetate is recommended instead of 0.3 M sodium acetate, since triphosphates are less likely to precipitate under these conditions. 2 M醋酸铵为三磷酸去除（或其他标记），而不是建议的0.3 M醋酸钠，因为三磷酸沉淀在这些情况下是不太可能的。 Ammonium acetate is not recommended if the nucleic acid sample will be 5 phosphorylated by T4 kinase or tailed at the 3 end with terminal transferase, since residual ammonium ions will inhibit these two enzymes.醋酸铵不建议，如果核酸样品将在5末端转移的磷酸化，T4激酶或在3尾“，因为残留的铵离子会抑制这两种酶。 Alternatively, LiCl can be used as the salt for precipitation.另外，氯化锂可以用作盐沉淀。 Instead of addition of 1/10 volume 3 M sodium acetate, add 1/10 volume 8 M LiCl.相反，另外1/10体积3 M醋酸钠，加1/10体积8米氯化锂。 Since LiCl is very soluble in ethanol, the resulting precipitate is relatively salt-free.由于氯化锂是非常易溶于乙醇，由此产生的沉淀物是相对无盐。 LiCl should be avoided, however, if precipitated RNA will be used as template for reverse transcription after precipitation.应避免使用氯化锂，然而，如果沉淀RNA将沉淀后的逆转录的模板。 Two Variations of the Precipitation Procedure降水过程的变化 If it is desirable to keep the volume of the precipitating nucleic acids to a minimum, isopropanol at a volume equal to the volume of the aqueous DNA solution can be substituted for the ethanol in the precipitation reaction.如果是可取保持沉淀核酸量到最低限度，在水溶液中的DNA溶液的体积等于体积的异丙醇，可以代替乙醇沉淀反应。 With this substitution, precipitation can be performed from a starting aqueous volume of 700 liL in a single microcentrifuge tube.这种替代，降水可以从700 LIL开始在一个单一的离心管中的水体积。 Isopropanol is not as volatile as ethanol, and is therefore more difficult to remove by evaporation in a vacuum centrifuge.异丙醇是不是如乙醇挥发，因此更难以去除在真空离心蒸发。 Some salts are less soluble in isopropanol, and may be precipitated with the nucleic acids.一些盐不溶于异丙醇中，并可能与核酸的沉淀。 It is recommended that isopropanol precipitation be followed immediately by a conventional ethanol precipitation to eliminate residual isopropanol and salt.据建议，随后立即由一个传统的乙醇沉淀，消除残留的异丙醇和盐，异丙醇沉淀。 Isolation of DNA基因组DNA提取 Because of the large size and the fragile nature of chromosomal DNA, it is unlikely that anyone has ever isolated it in an intact, undamaged form.由于庞大的规模和脆弱性染色体DNA，这是不可能的，任何人都曾经是孤立的，它在一个完整的，没有损坏的形式。 Several isolation procedures have been developed that provide DNA in a biologically active form, but this does not mean it is completely undamaged.几个隔离程序已开发提供DNA生物活性的形式，但这并不意味着它是完全没有损坏。 These DNA preparations are stable, of high molecular weight and relatively free of RNA and protein.这些DNA的筹备工作稳定，超高分子量和相对自由的RNA和蛋白质。 Here, a general method will be described for the isolation of DNA in a stable, biologically active form from microorganisms.在这里，一般的方法将在一个稳定的，具有生物活性的形式，从微生物的DNA提取。 The procedure outlined is applicable to many microorganisms and can be modified as necessary.所述的程序是适用于许多微生物，并可以根据需要进行修改。 Designing an isolation procedure for DNA requires extensive knowledge of the chemical stability of DNA as well as its condition in the cellular environment.设计DNA分离过程，需要广泛的知识蜂窝环境中的化学稳定性的DNA以及它的条件。 Several chemical bonds may be susceptible to cleavage during the extraction process.在提取过程中，几种化学债券可能受到切割。 The experimental factors that must be considered and their effects on various structural aspects of intact DNA are outlined below.实验必须考虑的因素和各种完整的DNA结构方面的影响概述如下。 1. 1。 pH pH值 (a) Hydrogen bonding between the complementary strands is stable between pH 4 and 10. （一）氢之间的互补链结合是稳定的pH值为4和10之间。 (b) The phosphodiester linkages in the DNA backbone are stable between pH 3 and 12. （二）在DNA骨架的磷酸二酯的联系是稳定的pH值3和12之间。 (c) N-glycoside bonds to purine bases (adenine and guanine) are hydrolyzed at pH values of 3 and less. （三）的N-糖苷债券嘌呤碱基（腺嘌呤和鸟嘌呤）水解的pH值和小于3。 2. 2。 Temperature温度 (a) There is considerable variation in the temperature stability of the hydrogen bonds in the double helix, but most DNA will begin to unwind in the range of 80-90C. （一）有相当大的变化，在温度稳定的双螺旋结构中的氢键，但大多数的DNA将开始放松范围在80-90C。 b. Phosphodiester linkages and N-glycoside bonds are stable up to IOOOC.磷酸的联系和稳定的N-糖苷键的高达IOOOC。 3. 3。 Ionic Strength离子强度 (a) DNA is most stable and soluble in salt solutions. （一）DNA是最稳定和可溶性盐溶液中。 Salt concentrations of less than 0.1 M weaken the hydrogen bonding between complementary strands.盐浓度小于0.1米削弱互补链之间的氢键。 4. 4。 Cellular Conditions细胞条件 (a) Before the DNA can be released, the bacterial cell wall must be lysed. （一）之前可以释放的DNA，细菌的细胞壁，必须裂解。 The ease with which the cell wall is disrupted varies from organism to organism.与细胞壁被破坏的易用性，不同机体有机体。 In some cases (yeast), extensive grinding or sonic treatment is required, whereas in others (B. subtilis), enzyme hydrolysis of the cell wall is possible.在某些情况下（酵母），需要广泛的研磨或超声波治疗，而在其他（枯草杆菌），细胞壁水解酶是可能的。 (b) Several enzymes are present in the cell that may act to degrade DNA, but the most serious damage is caused by the deoxyribonucleases. （二）一些酶可能采取行动，降解DNA的细胞中存在，但由deoxyribonucleases造成的损害最为严重。 These enzymes catalyze the hydrolysis of phosphodiester linkages.这些酶催化水解磷酸联系。 (c) Native DNA is present in the cell as DNA-protein complexes. （三）原住民的DNA是细胞DNA-蛋白质复合物。 The proteins (basic proteins called histones) must be dissociated during the extraction process.在提取过程中，必须是分离的蛋白质（蛋白质称为组蛋白的基本）。 5. 5。 Mechanical Stress on the DNA机械应力的DNA (a) Gentle manipulations may not always be possible during the isolation process. （一）温和的操作可能并不总是可能的隔离过程中。 Grinding, shaking, stirring, and other disruptive procedures may cause cleavage (shearing or scission) of the DNA chains.磨，震动，搅拌，和其他破坏性程序，可能会导致的DNA 链的断裂（剪切或断裂）。 This usually does not cause damage to the secondary structure of the DNA, but it does reduce the length of the molecules. 这通常不会造成损害的DNA的二级结构，但它确实降低分子的长度。 Now that these factors are understood, a general procedure of DNA extraction may be outlined:现在，这些因素是可以理解的，DNA提取的一般程序可概括： Step 1.第1步。 Disruption of the cell membrane and release of the DNA into a medium in which it is soluble and protected from degradation中断介质，它是降解可溶性和保护细胞膜和DNA的释放 The isolation procedure described here calls for the use of an enzyme, lysozyme, to disrupt the cell membrane.这里所描述的隔离程序要求使用的一种酶，溶菌酶，破坏细胞膜。 Lysozyme catalyzes the hydrolysis of glycosidic bonds in cell wall carbohydrates, thus causing destruction of the outer membrane and release of DNA and other cellular components.溶菌酶催化细胞壁碳水化合物糖苷键的水解，从而造成破坏DNA和其他细胞成分的外膜和释放。 The medium for solution of DNA is a buffered, saline solution containing EDTA. DNA溶液的介质是一个缓冲，含EDTA的盐溶液。 DNA, because it is ionic, is more soluble and stable in salt solution than in distilled water.的DNA，因为它是离子，是更多的可溶性盐溶液中的稳定比蒸馏水。 The EDTA serves at least two purposes. EDTA的服务至少有两个目的。 First, it binds divalent metal ions (Cd, Mg 2 +, Mn 2 +) that could form salts with the anionic phosphate groups of the DNA. Second, it inhibits deoxyribonucleases that have a requirement for Mg2+ or Mn 2 1. Citrate has occasionally been used as a chelating agent for DNA extraction; however, it is not an effective agent for binding Mn 2 . The mildly alkaline medium (pH 8) acts to reduce electrostatic interaction between DNA and the basic histones and the polycationic amines, spermine and spermidine (see Experiment 21). The relatively high pH also tends to diminish nuclease activity and denature other proteins.首先，它结合二价金属离子（镉“，镁，锰2 +），可能形成的DNA的阴离子磷酸盐组盐。其次，它抑制deoxyribonucleases要求为镁+ 或锰 2 1。 柠檬酸有偶尔被用作螯合剂DNA提取的，但它是不弱碱性介质中（pH值8）有效结合锰2“的代理行为，以减少DNA的基本组蛋白和阳离子胺，精胺之间的静电相互作用。和亚精胺（见实验21）。相对较高的pH值也趋于减少核酸酶的活性和变性其他蛋白质。 Step 2.第2步。 Dissociation of the protein-DNA complexes蛋白质-DNA复合物的解离 Detergents are used at this stage to disrupt the ionic interactions between positively charged histones and the negatively charged backbone of DNA.在这个阶段用于洗涤剂，扰乱离子带正电荷的组蛋白和带负电荷的DNA骨干之间的相互作用。 Sodium dodecyl sulfate (SDS), an anionic detergent, binds to proteins and gives them extensive anionic character.十二烷基硫酸钠（SDS），阴离子洗涤剂，蛋白质结合，并为他们提供了广泛的阴离子字符。 A secondary action of SDS is to act as a denaturant of deoxyribonucleases and other proteins.二次行动的SDS是作为一个deoxyribonucleases和其他蛋白质的变性。 Also favoring dissociation of protein-DNA complexes is the alkaline pH, which reduces the positive character of the histones.也有利于蛋白质-DNA复合物的分离，是碱性的pH值，从而降低了组蛋白的正面人物。 To ensure complete dissociation of the DNA-protein complex and to remove bound cationic amines, a high concentration of a salt (NaCl or sodium perchlorate) is added.补充，以确保完整的DNA-蛋白质复合体分离和删除绑定阳离子胺，高浓度的盐（氯化钠或氯酸钠）。 The salt acts by diminishing the ionic interactions between DNA and cations.盐的行为，减少DNA和阳离子之间的离