DNA的溶解度问题Word格式文档下载.docx

1、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

2、 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 therefo

3、re 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-）

4、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 CH3CO

5、O-.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 electrostati

6、c 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 c

7、ome 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

8、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 Clon

9、ing, 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

10、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 precipit

11、ations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

12、.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 re

13、verse 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 a

14、s 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

15、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 One of the most commonly used and useful methods for isolation and concentration of DNA and RNA from aqueous solutions is phenol/chloroform extr

16、action 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

17、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

18、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 denatura

19、tion 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

20、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

21、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, multi

22、ple 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 with

23、stand 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 rec

24、ommended 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 nucleotid

25、es 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 quant

26、itative.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

27、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 p

28、roblem, 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

29、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

30、 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