DNA的溶解度問(wèn)題

1、乙醇沉淀法是一種常用的技術(shù)集中的鹽在水溶液中的核酸（DNA或RNA）準(zhǔn)備。 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，加入鹽和乙醇水溶液，這迫使核酸沉淀出的解決方案。 The precipitated nucleic acid can then be separated from the rest of the solution by cent

2、rifugation.然后可以休息的解決方案，通過(guò)離心分離沉淀核酸。 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乙醇洗滌沉淀，然后再經(jīng)過(guò)離心步驟乙醇被刪除，并允許核酸顆粒干燥，然后在干凈的緩沖溶液中懸浮。 So how does

3、 this work?那么如何工作的呢？ A bit about solubility 關(guān)于溶解度的位. First we need to know why nucleic acids are soluble in water.首先，我們需要知道為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 a

4、toms (see the diagram on the right).水是極性分子 - 它有一個(gè)部分負(fù)電荷的氧原子附近，由于共用電子對(duì)，部分正電荷的氫原子（見右圖）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.極性分子，如DNA或RNA，因?yàn)檫@些費(fèi)用，可以與水分子的靜電相互作用，使他們能夠很容易溶解于水。 Polar

5、 molecules can therefore be described as hydrophilic and non-polar molecules, which can't easily interact with water molecules, are hydrophobic.極性分子，因此可以被描述為親水性和非極性分子，不能輕易與水分子相互作用，疏水。 Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar phosphate ba

6、ckbone.核酸是親水性由于帶負(fù)電荷的磷（PO3-）組沿糖磷酸骨架。 The role of the salt 鹽的作用. Ok, so back to the protocol.好吧，這樣的協(xié)議。 The role of the salt in the protocol is to neutralize the charges on the sugar phosphate backbone.鹽在協(xié)議中的作用是消除糖磷酸骨架上的收費(fèi)。 A commonly used salt is sodium acetate.一種常用的鹽是醋酸鈉。 In solution, sodium acetate

7、breaks up into Na+ and CH3COO-.在溶液中，分解成Na +和醋酸 - 醋酸鈉。 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.帶正電的鈉離子中和負(fù)電荷的核酸PO3組，分子遠(yuǎn)低于親水性，因此多不溶于水。 The role of the et

8、hanol 乙醇的作用. The electrostatic attraction between the Na+ ions in solution and the PO3- ions are dictated by Coulomb's Law , which is affected by the dielectric constant of the solution.鈉離子在溶液中和PO3-離子之間的靜電吸引力， 庫(kù)侖定律 ，這是解決方案的電介質(zhì)常數(shù)的影響決定。 Water has a high dielectric constant, which makes it fairly d

9、ifficult for the Na+ and PO3- to come together.水具有較高的介電常數(shù)，這使得它相當(dāng)困難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 it's charge and make the nucleic acid less hydrophilic, causing it to drop out of solut

10、ion.另一方面乙醇低得多的介電常數(shù)，使得它的Na +更容易互動(dòng)，PO3-，它的電荷屏蔽，使核酸的親水性，使其下降了解決方案。 The role of temperature 溫度的作用. Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (eg -20 or -80C) is commonly cited in protocols as necessary in protocols.作為必要的協(xié)議，在協(xié)議中普遍提及的核酸/鹽/乙醇的混合物在低溫（如-20或-80C）的孵化。 However, acc

11、ording 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，根據(jù)，曼尼阿蒂斯等 （分子克隆實(shí)驗(yàn)手冊(cè)第二

12、版.第二版- ？！我需要得到一個(gè)新的版本），這是不是必需的，因?yàn)?0ng/mL低濃度核酸沉淀在0-4C，所以在冰上潛伏期為15-30分鐘就足夠了。 The wash step with 70% ethanol 用70乙醇洗滌步驟. This step is to wash any residual salt away from the pelleted DNA.這一步是洗任何剩余的鹽顆粒的DNA。 A few tips on nucleic acid precipitation 核酸沉淀的一些技巧. § Choice of salt 鹽的選擇 § Use Sodium ac

13、etate (0.3M final conc, pH 5.2) for routine DNA precipitations使用常規(guī)的DNA沉淀醋酸鈉 （0.3M終濃度，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 won't precipitate with the DNA.使用DNA含有SDS自氯化鈉保持SDS在70乙醇中易溶，所以它不會(huì)沉淀的DNA樣本， 氯化鈉

14、 （0,2米決賽濃度）。 § Use Lithium Chloride (0.8M final conc) for RNA.使用氯化鋰 （0.8M終濃度）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

15、 polymerase so LiCl is no good for RNA preps for in vitro translation or reverse transcription.這是因?yàn)?.5-3乙醇卷應(yīng)該被用于RNA沉淀和LiCl是比乙酸鈉溶于乙醇，所以不會(huì)沉淀，但要小心 - 氯離子會(huì)抑制蛋白質(zhì)的合成和DNA聚合酶，使氯化鋰的RNA PREPS沒有好的在體外翻譯或反轉(zhuǎn)錄。 In these cases, use NaAc.在這種情況下，使用醋酸鈉。 § Use Ammonium acetate (2M final conc) for the removal of dNT

16、Ps, but do not use for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme.用于去除dNTPs濃度的醋酸銨 （2M終濃度），但不使用T4聚核苷酸激酶反應(yīng)制備的DNA銨離子抑制酶。 § To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides) 在低濃度或小

17、核酸件（少于100個(gè)核苷酸）的降水以增加產(chǎn)量 § Add MgCl2 to a final concentration of 0.01M氯化鎂的終濃度為0.01M § Increase the time of incubation ice before centrifugation to 1 hour.潛伏期冰離心前的時(shí)間增加至1小時(shí)。 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 o

18、f 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，從水溶液中的DNA和RNA的分離和濃度的方法之一是酚/氯仿抽提，乙醇沉淀。 During organic extraction, protein contaminants are denatured and partiti

19、on either with the organic phase or at the interface between organic and aqueous phases, while nucleic acids remain in the aqueous phase.在有機(jī)萃取，蛋白質(zhì)污染物變性和分區(qū)與有機(jī)相或有機(jī)相和水相之間的界面，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.在這

20、個(gè)協(xié)議中使用的苯酚緩沖，以防止破壞核酸在苯酚氧化的產(chǎn)品。 Be aware that phenol can cause severe chemical burns on skin and will damage clothing.要知道，酚可引起嚴(yán)重的化學(xué)燒傷，皮膚上，會(huì)損傷衣物。 Wear gloves, safety glasses, and a laboratory coat when working with phenol.與苯酚工作時(shí)戴手套，安全眼鏡，實(shí)驗(yàn)室大衣。 In the method presented here, phenol/chloroform (50%/50%; v/

21、v) is recommended for extraction.建議在這里提出的方法，酚/氯仿（50/ 50V / V） 提取 。 In most cases, this mixture provide在大多數(shù)情況下，這種混合物提供 good protein denaturation and a tighter interphase between the aqueous and organic phases. 良好的蛋白質(zhì)變性和水相和有機(jī)相之間的更緊密相間。 If there is a problem with excessive foaming during the extraction

22、, isoamyl alcohol can be added to obtain an organic composition of phenol/chloroform (50%/49%)/isoamyl alcohol (I%). 提取過(guò)程中過(guò)多的泡沫如果有一個(gè)問(wèn)題，異戊醇可以增加獲得的酚/氯仿（50/ 49）/異戊醇（）的有機(jī)組成。 Conventional Ethanol Precipitation傳統(tǒng)的乙醇沉淀 During the ethanol precipitation, salts and other solutes such as residual phenol and ch

23、loroform remain in solution while nucleic acids form a white precipitate that can,easily be collected by centrifugation.在乙醇沉淀，鹽和其他溶質(zhì)，如殘余苯酚和氯仿溶液中保持，而核酸形成白色沉淀物，可以很容易地通過(guò)離心收集。 If the aqueous volume is less than 450 pL, the reaction can be performed in a microcentrifuge tube.如果水的體積小于450 PL，反應(yīng)可在離心管中。 This

24、 is the most convenient format for performing organic extractions and ethanol precipitations.這是最方便的格式進(jìn)行有機(jī)提取物和乙醇沉淀。 For larger volumes, multiple microcentrifuge tubes can be used, or the reaction can be scaled up.對(duì)于體積較大，可以使用多種離心管，可以擴(kuò)展或反應(yīng)。 When scaling the reaction up, use tightly capped polypropylene

25、 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 fo

26、r 15 min at 4 OC.當(dāng)縮放的反應(yīng)了，使用的酚/氯仿提取，并離心機(jī)蓋緊聚丙烯管在2500在室溫下轉(zhuǎn)速來(lái)解決階段聚苯乙烯管能不能承受的酚/氯仿乙醇沉淀可以將在15個(gè)執(zhí)行- ?；?0 -毫升的Corex管，沉淀15分鐘，在4離心收集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 f

27、or 20 min.據(jù)建議，Corex公司管是使用在 50硝酸浸泡在蒸餾水徹底清洗，高壓滅菌20分鐘，1小時(shí)前洗酸。 In our hands, nucleic acid fragments and oligonucleotides longer than 15 nucleotides can be efficiently precipitated using this protocol.在我們的手，核酸片段和寡核苷酸超過(guò)15個(gè)核苷酸可以有效地沉淀，使用此協(xié)議。 For efficient precipitation, the nucleic acid concentration should

28、 be at least 10 gg/mL.為了有效降水，核酸濃度應(yīng)該是至少10 GG /毫升。 Lower concentrations of nucleic acids can be precipitated, but the recovery may not be quantitative.可以沉淀核酸的濃度較低，但經(jīng)濟(jì)復(fù)蘇可能不定量。 To precipitate lower nucleic acid concentrations, incubate the precipitate at -20 'C (or on dry ice) for 4 hr to overnight,

29、 and centrifuge for 30 min to collect the precipitate.沉淀核酸濃度較低，在-20“C”（或干冰）孵育4小時(shí)至過(guò)夜，離心30分鐘，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

30、extraction and precipitation procedure.另外，可以有效地沉淀核酸納克數(shù)量加入酵母tRNA載波解決方案前發(fā)起的提取和降水過(guò)程，獲得了10克/毫升的核酸濃度。 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 po

31、lynucleotide kinase). tRNA的載體的存在通常是沒有問(wèn)題的，除承運(yùn)人時(shí)，會(huì)干擾隨后的酶樣品的操作（例如，如果將DNA片段，用T4多核苷酸激酶末端標(biāo)記）。 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 precipitat

32、e with the nucleic acid sample.這種方法最常規(guī)應(yīng)用的建議鹽0.3 M醋酸鈉（終濃度），這是更大于0.3 m氯化鈉溶于乙醇，因此不太可能與核酸樣品沉淀。 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.對(duì)于含有十二烷基硫酸鈉（SDS）的樣品，建議的鹽是氯化鈉0.2 mol以來(lái)，SDS是在這些條件下易溶

33、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醋酸銨為三磷酸去除（或其他標(biāo)記），而不是建議的0.3 M醋酸鈉，因?yàn)槿姿岢恋碓谶@些情況下是不太可能的。 Ammonium acetate is not recommended if the

34、 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.醋酸銨不建議，如果核酸樣品將在5末端轉(zhuǎn)移的磷酸化，T4激酶或在3尾“，因?yàn)闅埩舻匿@離子會(huì)抑制這兩種酶。 Alternatively, LiCl can be used as the salt for precipitation.另外，氯化鋰可

35、以用作鹽沉淀。 Instead of addition of 1/10 volume 3 M sodium acetate, add 1/10 volume 8 M LiCl.相反，另外1/10體積3 M醋酸鈉，加1/10體積8米氯化鋰。 Since LiCl is very soluble in ethanol, the resulting precipitate is relatively salt-free.由于氯化鋰是非常易溶于乙醇，由此產(chǎn)生的沉淀物是相對(duì)無(wú)鹽。 LiCl should be avoided, however, if precipitated RNA will be u

36、sed as template for reverse transcription after precipitation.應(yīng)避免使用氯化鋰，然而，如果沉淀RNA將沉淀后的逆轉(zhuǎn)錄的模板。 Two Variations of the Precipitation Procedure降水過(guò)程的變化 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 sol

37、ution 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溶液的體積等于體積的異丙醇，可以代替乙醇沉淀反應(yīng)。 With this substitution, precipitation can be performed from a starting aqueous volume of 700 liL in a single microcentrifuge tube.這種替代，降水可以從700 LIL開始在一個(gè)單一的離心管中的水體積。 Isopropanol

38、 is not as volatile as ethanol, and is therefore more difficult to remove by evaporation in a vacuum centrifuge.異丙醇是不是如乙醇揮發(fā)，因此更難以去除在真空離心蒸發(fā)。 Some salts are less soluble in isopropanol, and may be precipitated with the nucleic acids.一些鹽不溶于異丙醇中，并可能與核酸的沉淀。 It is recommended that isopropanol precipitatio

39、n be followed immediately by a conventional ethanol precipitation to eliminate residual isopropanol and salt.據(jù)建議，隨后立即由一個(gè)傳統(tǒng)的乙醇沉淀，消除殘留的異丙醇和鹽，異丙醇沉淀。 Isolation of DNA基因組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

40、, undamaged form.由于龐大的規(guī)模和脆弱性染色體DNA，這是不可能的，任何人都曾經(jīng)是孤立的，它在一個(gè)完整的，沒有損壞的形式。 Several isolation procedures have been developed that provide DNA in a biologically active form, but this does not mean it is completely undamaged.幾個(gè)隔離程序已開發(fā)提供DNA生物活性的形式，但這并不意味著它是完全沒有損壞。 These DNA preparations are stable, of high m

41、olecular weight and relatively free of RNA and protein.這些DNA的籌備工作穩(wěn)定，超高分子量和相對(duì)自由的RNA和蛋白質(zhì)。 Here, a general method will be described for the isolation of DNA in a stable, biologically active form from microorganisms.在這里，一般的方法將在一個(gè)穩(wěn)定的，具有生物活性的形式，從微生物的DNA提取。 The procedure outlined is applicable to many micr

42、oorganisms and can be modified as necessary.所述的程序是適用于許多微生物，并可以根據(jù)需要進(jìn)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.設(shè)計(jì)DNA分離過(guò)程，需要廣泛的知識(shí)蜂窩環(huán)境中的化學(xué)穩(wěn)定性的DNA以及它的條件。 Several chemical bonds may be su

43、sceptible to cleavage during the extraction process.在提取過(guò)程中，幾種化學(xué)債券可能受到切割。 The experimental factors that must be considered and their effects on various structural aspects of intact DNA are outlined below.實(shí)驗(yàn)必須考慮的因素和各種完整的DNA結(jié)構(gòu)方面的影響概述如下。 1. 1。 pH pH值 (a) Hydrogen bonding between the complementary strand

44、s is stable between pH 4 and 10. （一）氫之間的互補(bǔ)鏈結(jié)合是穩(wěn)定的pH值為4和10之間。 (b) The phosphodiester linkages in the DNA backbone are stable between pH 3 and 12. （二）在DNA骨架的磷酸二酯的聯(lián)系是穩(wěn)定的pH值3和12之間。 (c) N-glycoside bonds to purine bases (adenine and guanine) are hydrolyzed at pH values of 3 and less. （三）的N-糖苷債券嘌呤堿基（腺嘌呤和鳥

45、嘌呤）水解的pH值和小于3。 2. 2。 Temperature溫度 (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 80-90'C. （一）有相當(dāng)大的變化，在溫度穩(wěn)定的雙螺旋結(jié)構(gòu)中的氫鍵，但大多數(shù)的DNA將開始放松范圍在80-90'C。 b. Phosphodiester linkages and N-glyco

46、side bonds are stable up to IOOOC.磷酸的聯(lián)系和穩(wěn)定的N-糖苷鍵的高達(dá)IOOOC。 3. 3。 Ionic Strength離子強(qiáng)度 (a) DNA is most stable and soluble in salt solutions. （一）DNA是最穩(wěn)定和可溶性鹽溶液中。 Salt concentrations of less than 0.1 M weaken the hydrogen bonding between complementary strands.鹽濃度小于0.1米削弱互補(bǔ)鏈之間的氫鍵。 4. 4。 Cellular Conditions

47、細(xì)胞條件 (a) Before the DNA can be released, the bacterial cell wall must be lysed. （一）之前可以釋放的DNA，細(xì)菌的細(xì)胞壁，必須裂解。 The ease with which the cell wall is disrupted varies from organism to organism.與細(xì)胞壁被破壞的易用性，不同機(jī)體有機(jī)體。 In some cases (yeast), extensive grinding or sonic treatment is required, whereas in others

48、(B. subtilis), enzyme hydrolysis of the cell wall is possible.在某些情況下（酵母），需要廣泛的研磨或超聲波治療，而在其他（枯草桿菌），細(xì)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）一些酶可能采取行動(dòng)，降解DNA的細(xì)胞中存在，但由deoxyribonucleases造成的損害最為

49、嚴(yán)重。 These enzymes catalyze the hydrolysis of phosphodiester linkages.這些酶催化水解磷酸聯(lián)系。 (c) Native DNA is present in the cell as DNA-protein complexes. （三）原住民的DNA是細(xì)胞DNA-蛋白質(zhì)復(fù)合物。 The proteins (basic proteins called histones) must be dissociated during the extraction process.在提取過(guò)程中，必須是分離的蛋白質(zhì)（蛋白質(zhì)稱為組蛋白的基本）。 5.

50、 5。 Mechanical Stress on the DNA機(jī)械應(yīng)力的DNA (a) Gentle manipulations may not always be possible during the isolation process. （一）溫和的操作可能并不總是可能的隔離過(guò)程中。 Grinding, shaking, stirring, and other disruptive procedures may cause cleavage (shearing or scission) of the DNA chains.磨，震動(dòng)，攪拌，和其他破壞性程序，可能會(huì)導(dǎo)致的DNA 鏈的斷裂（

51、剪切或斷裂）。 This usually does not cause damage to the secondary structure of the DNA, but it does reduce the length of the molecules. 這通常不會(huì)造成損害的DNA的二級(jí)結(jié)構(gòu)，但它確實(shí)降低分子的長(zhǎng)度。 Now that these factors are understood, a general procedure of DNA extraction may be outlined:現(xiàn)在，這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。 Disr

52、uption of the cell membrane and release of the DNA into a medium in which it is soluble and protected from degradation中斷介質(zhì)，它是降解可溶性和保護(hù)細(xì)胞膜和DNA的釋放 The isolation procedure described here calls for the use of an enzyme, lysozyme, to disrupt the cell membrane.這里所描述的隔離程序要求使用的一種酶，溶菌酶，破壞細(xì)胞膜。 Lysozyme catalyz

53、es the hydrolysis of glycosidic bonds in cell wall carbohydrates, thus causing destruction of the outer membrane and release of DNA and other cellular components.溶菌酶催化細(xì)胞壁碳水化合物糖苷鍵的水解，從而造成破壞DNA和其他細(xì)胞成分的外膜和釋放。 The medium for solution of DNA is a buffered, saline solution containing EDTA. DNA溶液的介質(zhì)是一個(gè)緩沖，含

54、EDTA的鹽溶液。 DNA, because it is ionic, is more soluble and stable in salt solution than in distilled water.的DNA，因?yàn)樗请x子，是更多的可溶性鹽溶液中的穩(wěn)定比蒸餾水。 The EDTA serves at least two purposes. EDTA的服務(wù)至少有兩個(gè)目的。 First, it binds divalent metal ions (Cd", Mg 2 +, Mn 2 +) that could form salts with the anionic phospha

55、te 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 electros

56、tatic 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，它結(jié)合二價(jià)金屬離子（鎘“，鎂，錳2 +），可能形成的DNA的陰離子磷酸鹽組鹽。其次，它抑制deoxyribonucleases要求為鎂+ 或錳 2 1。 檸

57、檬酸有偶爾被用作螯合劑DNA提取的，但它是不弱堿性介質(zhì)中（pH值8）有效結(jié)合錳2“的代理行為，以減少DNA的基本組蛋白和陽(yáng)離子胺，精胺之間的靜電相互作用。和亞精胺（見實(shí)驗(yàn)21）。相對(duì)較高的pH值也趨于減少核酸酶的活性和變性其他蛋白質(zhì)。 Step 2.第2步。 Dissociation of the protein-DNA complexes蛋白質(zhì)-DNA復(fù)合物的解離 Detergents are used at this stage to disrupt the ionic interactions between positively charged histones and the neg

58、atively charged backbone of DNA.在這個(gè)階段用于洗滌劑，擾亂離子帶正電荷的組蛋白和帶負(fù)電荷的DNA骨干之間的相互作用。 Sodium dodecyl sulfate (SDS), an anionic detergent, binds to proteins and gives them extensive anionic character.十二烷基硫酸鈉（SDS），陰離子洗滌劑，蛋白質(zhì)結(jié)合，并為他們提供了廣泛的陰離子字符。 A secondary action of SDS is to act as a denaturant of deoxyribonucle

59、ases and other proteins.二次行動(dòng)的SDS是作為一個(gè)deoxyribonucleases和其他蛋白質(zhì)的變性。 Also favoring dissociation of protein-DNA complexes is the alkaline pH, which reduces the positive character of the histones.也有利于蛋白質(zhì)-DNA復(fù)合物的分離，是堿性的pH值，從而降低了組蛋白的正面人物。 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.補(bǔ)充，以確保完整的DNA-蛋白質(zhì)復(fù)合體分離和刪除綁定陽(yáng)離子胺，高濃度的鹽（氯化鈉或氯酸鈉）。 The salt acts by diminishing the ionic interactions between DNA and cations.鹽的行為，減少DNA和陽(yáng)離子之間的