RNA的变性电泳原理

RNA的变性电泳原理 RNA的琼脂糖凝胶电泳 实验原理 RNA电泳可以在变性及非变性两种条件下进行。非变性电泳使用1.0%--1.4%的凝胶，不同的RNA条带也能分开，但无法判断其分子量。只有在完全变性的条件下，RNA的泳动率才与分子量的对数呈线性关系。因此要测定RNA分子量时，一定要用变性凝胶。在需快速所提总RNA样品完整性时，配制普通的1%琼脂糖凝胶即可。 基本过程同DNA电泳一样，但应明确一点的是，因为RNA分子对RNA酶的作用非常敏感，因此必须用对RNA酶有抑制作用DEPC水来配置所有溶液，所有与RNA接触的仪器和装置都要严格处理以尽量减少RNA酶对样品的降解;另外，因为RNA分子有二、三级结构可以影响其电泳结果，因此电泳时应在变性剂存在下进行，常用的变性剂为甲醛和戊二醛。 RNA非变性琼脂糖凝胶检测 实验材料、器具及药品 蘑菇的总RNA溶液。 电泳仪，电泳槽，电子天平，移液器，枪头，微波炉，紫外透射检测仪等。 琼脂糖，1XTAE电泳缓冲液，0.5μg/ml溴化乙锭(EB)10X载样缓冲液。 实验步骤 (1)用1×TAE电泳缓冲液制作琼脂糖凝胶，加1×TAE电泳缓冲液至液面覆盖凝胶。 (2)在超净工作台上，用移液器吸取总RNA样品4 ul于封口膜上。在实验台上再加入5 ul 1×TAE电泳缓冲液及1 ul 的10X载样缓冲液，混匀后，小心加入点样孔。 (3)打开电源开关，调节电压至100V，使RNA由负极向正极电泳，约30min后将凝胶放入EB染液中染色5min,用清水稍微漂洗。在紫外透射检测仪上观察RNA电泳结果。influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there 非变性电泳:上样量超过 3ug，电压超过 6V/cm，电泳缓冲液时间太长，均可能导致 28S 和 18S 条带分不开。使用 2ug 上样量，电压小于 6V/cm，使用新鲜的电泳缓冲液并且频繁混匀两极的缓冲液，是获得好的电泳结果的前提。(以 DNA 标准为参照，28S 和 18S 分别位于 2.0kb 和 0.9kb 左右。) 变性电泳条带变淡:EB 与单链的结合能力要差一些，故同样的上样量，变性电泳比非变性电泳要淡一些。另外的可能是甲醛的质量不高。 RNA的变性琼脂糖凝胶检测 试剂: (1) MOPS缓冲液(10*):0.4mol/L 吗啉代丙烷磺酸(MOPS)(PH7.0)，0.1mol/L NaAc, 10mol/L EDTA。 (2)上样染料:50%甘油，1mmol/L EDTA ,0.4%溴酚蓝，0.4%二甲苯蓝。 (3)甲醛。 (4)去离子甲酰胺。 influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there 电泳槽清洗:去污剂洗干净(一般浸泡过夜)——水冲洗——乙醇干燥——3%H2O2 灌满——室温放置10分钟——0.1%DEPC水冲洗。 操作: (1) 将制胶用具用70%乙醇冲冼一遍，晾干备用。 (2) 配制琼脂糖凝胶。 ?称取0.5g琼脂糖，置干净的100ml锥形瓶中，加入40ml蒸馏水，微波炉内加热使 琼脂糖彻底溶化均匀。 ?待胶凉至60--70 ?，依次向其中加入9ml甲醛、5ml 10X MOPS缓冲液和0.5 ul溴 化乙锭，混合均匀。 ?灌制琼脂糖凝胶。 (3) 样品准备: ? 取DEPC处理过的500 ul小离心管，依次加入如下试剂: 10x MOPS缓冲液2ul， 甲醛3.5 ul,甲酰胺(去离子)10 ul，RNA 样品4.5 ul,混匀。 ? 将离心管置于60?水浴中保10分钟，再置冰上2分钟。 ? 向管中加入3 ul上样染料，混匀。 (4)上样。 (5)电泳:电泳槽内加入1XMOPS缓冲液，于7.5V/ml 的电压下电泳。 (6)电泳结束后，在紫外灯下检查结果。 influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there RNA提取、电泳注意事项，有几点说得很好啊 快速的操作，低温环境，少量取上清 主要是抽提之后吸取上清时要特别小心，只要不把蛋白层吸出来就不会有RNase污染。 分子克隆第二版343页,上关于提取RNA所用的玻璃容器的处理方法,是用前180度干烤8小时或更长时间;另一种方法是0.1%的depc水浸泡(37度2小时),然后灭菌水淋洗数次,100度干烤15分钟,然后高压灭菌除去depc.我们实验室一直是用170度考4小时,且不准离人,不准过夜,可能是怕 烤箱长时间高温,线路老化会发生危险吧 提取RNA所用的枪头，EP管。直接高压烘干即可。如果用0.1,DEPC处理，要在DEPC配置后振摇1小时后再浸泡枪头，EP管方有效。我做的都是将枪头泡在配制好的depc中，摇influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there 振过夜，然后倒掉depc注意要到干净，再高压，为了防止仍残留液体可120度干烤一会 A.对于提取RNA来说,我认为关键的一点在于实验器材的准备.包括从最初的标本的制备,保存,以及之后的试剂的准备,匀浆器,镊子,tip头的去除RNase处理,尤其是去除RNase的DEPC处理步骤 B. 您在处死大鼠前,先准备好干净的冻存管(用1.5ml的Ep管也可以,就是后面您在从液氮中取出的时候容易爆,您的注意安全),然后将去除的骨骼肌立即用 干净的剪刀分成半颗到一颗黄豆大小,装入冻存管后立即投入液氮保存.个人认为在这里使用装标本的管子没必要用DEPC处理 C.在提取RNA之前,将试剂(TRIzol)和器械(tip头,镊子,匀浆器)都准备好 D.从液氮取出标本后,立即转移进入事先加入TRIzol的匀浆器内(最好置冰上操作),立即匀浆,一直到标本完全碾磨碎,这个时候TRIzol液体成浑浊状,而且匀浆器的壁上没有太多的黏附组织,然后将TRIzol转出,继续后续的步骤. 1 RNase是一种蛋白酶，能够降解RNA。 2我们的手上皮肤上有很多，应该是对我们的身体起保护作用。保护不被RNA病毒感染,或者什么的。 3 这个酶高效稳定。要么在DEPC水中处理n小时，要么在160度高温烘烤6小时。此酶才会失效。 4 所谓DEPC，是一种叫做焦碳酸二乙酯的东西。有毒啊。所谓DEPC水，一般指两种状态，a，配制好未消毒;b，配制好已消毒。通过高压消毒，该物质会降解，从而无毒。 1 无菌蒸馏水中加入0.1%(v/v)焦碳酸二乙酯(DEPC)，室温搅拌4小时以上至完全溶解，高压灭菌20分钟，冷却备用。这个是DEPC水的b状态。 2 如果你要用DEPC水处理Tips等等东东，那么就要用高压前的水，即DEPC水的a状态。把枪头管子等完全浸泡至少8小时，我一般都是过夜的，或者平时就泡在里面备用。 RNA 提取必须创造无RNase的环境，所有仪器与试剂均按照以下方法进行处理 :研钵、研棒、药匙、玻璃器皿等需在180?烘干4h以上; 电泳槽、胶板、梳子、用0.1 %,0.2%DEPC水处理过夜或者用洗涤灵清洗干净后用0.5%的SDS (Sodium dodecyl sulfate，抑制RNase的活性)浸泡过夜，然后用ddH2O冲洗干净，晾干备用;离心管，枪头等塑料器皿要用0.1%,0.2%DEPC水处理之 后(DEPC有致癌之嫌，须小心操作)。37?保温12h以上，然后高压灭菌，灭活DEPC ( Diethypyrocarbonate );溶液都需用经DEPC处理过的水配置， RNA提取的所有操作应尽可能在超净工作台上进行。 我们研究所里提RNA用的枪头和EP管都是高压两遍的，没有用DEPC水处理。 1、 有灭菌过的DEPC水，这一般是用来配75%乙醇用，因为乙醇若高压灭菌会挥发，所以乙醇是新开封专用的。 2、 没有经高压灭菌的DEPC水，这主要是用于配你提RNA所用的其他试剂(所说的试剂是可以经高压灭菌的)，总的来说，都得经过高压灭菌，目的就是要去除DEPC，以防止它以随后实验的干扰。 3、 DEPC处理水:无菌蒸馏水中加入0.1%(v/v)焦碳酸二乙酯(DEPC)，室温搅拌4小时以上，121 度高压灭菌20分钟，冷却备用。 凡是不能用高温烘烤的材料如塑料容器等皆可用0.1%的焦碳酸二乙酯(DEPC)水溶液处理,再influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there 用蒸馏水冲净。DEPC是RNA酶的化学修饰剂,它和RNA 酶的活性基团组氨酸的咪唑环反应而抑制酶活性。DEPC与氨水溶液混合会产生致癌物,因而使用时需小心。试验所用试剂也可用DEPC处理,加入DEPC至 0.1%浓度,然后剧烈振荡10分钟,再煮沸15分钟或高压灭菌以消除残存的DEPC,否则DEPC也能和腺嘌呤作用而破坏mRNA活性。但DEPC能与胺和巯基反应,因而含Tris和DTT的试剂不能用DEPC处理。Tris溶液可用DEPC处理的水配制然后高压灭菌。配制的溶液如不能高压灭菌,可用 DEPC处理水配制,并尽可能用未曾开封的试剂. 为啥在28s之后还是有影影约约的片断,和模糊的smear, 这都与非变性电泳方式有关。RNA的电泳，严格讲是要使用变性电泳的，我们平时所知道的 RNA 电泳的知识，实际上都是由变性电泳获得的。但因为非变性电泳实在是太方便了，同时，就一般检测而言，完全可以替代变性电泳，所以我们日常检测就都使用非变性电泳了。 电泳时间太长了容易产生降解。最好是在120V左右。操作的时候有没有注意戴口罩和手套，注意，手套要经常换，不要太节约了。那样也会影响RNA的质量。无论你是提什么RNA只要是用异丙醇沉淀的，都得用75,的酒精洗涤一次。 1 样品不要太多，抽提务必充分，室温放置5min; 2 200ul氯仿 剧烈振荡20s，室温放置2,15min; 3 13000g 离心 15min; 4 取上层水相,一般400-450ul就足够了，千万不要贪多～*** 5 加入500ul异丙醇 摇匀，室温放置10min; 6 12000g 离心 10min; 7 75,乙醇 1000ul 洗涤沉淀; 8 7500g 离心 5min;(12000转速太高了吧，沉淀不容易溶解的) 9 弃乙醇，吸干净残余的微量乙醇， 室温干燥3,5min;(时间太久沉淀不易溶解) 10 适量DEPC水溶解沉淀。 电泳的上样量1ug，电泳buffer用DEPC处理的水配制，电泳时间不要太久，95V，10,15min足够了。 建议跑RNA电泳。先用2,琼脂糖胶，若分离效果不好，可用甲醛变性胶。在上样缓冲液中注意加入RNA酶抑制剂。注意观察带形，质量较好的RNA亮度 。28S:18S应为2:1。还要注意观察，在加样孔或刚出加样孔附近，有没有带， 若有，可能为基因组DNA污染。 OD值只有1.5，可能为基因组DNA污染。参考一下分子克隆3。上面有OD值与污染DNA的关系。建议，酚氯仿抽提后，上清可少吸一些。提取后的RNA样品可用无RNA酶的DNA酶消化一下。注意该酶的buffer中有一种物质在 OD260有吸光度，应严格按照其protocol操作，减少误差。 只要用DEPC处理过的水，打开一瓶新的无水乙醇(或者专用于RNA的， 即只用处理过的枪头取过乙醇的)配就可以了。DEPC高温高压时变成CO2和水，再加上乙醇也很容易气化，可能是因为有气体产生吧，如果盖子太紧容易发生瓶子破掉的事情。(我还从来没有听说过把乙醇高温灭菌过)而且，湿热灭菌本身不足以去除RNA酶，所以我觉得有时候，灭菌可能反而引入RNA酶污染也不一 定哦 75%的乙醇用于提取RNA 时最好还是现用现配，乙醇最好是新的且以后不要用于其他方面，也就是留一瓶专门用于提取RNA。DEPC一般高压灭菌30分钟就可以了～ influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there 75%乙醇:灭菌后的千分之一DEPC水25ml 75ml无水乙醇.配好后装入高温烘烤的玻璃瓶中(160干烘四小时)，或塑料器皿的用千分之一的DEPC泡12小时以上再灭30分钟，存放于低温冰箱4度保存!!! 实验十一 动物组织细胞总RNA的提取 一、实验原理 RNA是基因达的中间产物，存在于细胞质与核中。对RNA进行操作在分子生物学中占有重要地位。获得高纯度和完整的RNA 是很多分子生物学实验所必需的，如Northern杂交、cDNA合成及体外翻译等实验的成败，在很大程度上取决于RNA的质量。由于细胞内的大部分RNA是以核蛋白复合体的形式存在，所以在提取RNA时要利用高浓度的蛋白质变性剂，迅速破坏细胞结构，使核蛋白与RNA分离，释放出RNA。再通过酚、氯仿等有机溶剂处理、离心，使RNA与其他细胞组分分离，得到纯化的总RNA。在提取的过程中要抑制内源和外源的RNase活性，保护RNA分子不被降解。因此提取必须在无RNase的环境中进行。可使用RNase抑制剂，如DEPC是RNase的强抑制剂，常用来抑制外源RNase活性。提取缓冲液中一般含SDS、酚、氯仿、胍盐等蛋白质变性剂，也能抑制RNase活性。并有助于除去非核酸成分。 本实验介绍异硫氰酸胍法和TRIzol法提取动物组织总RNA并通过电泳 进行鉴定。 二、仪器和试剂 1.仪器: 超净工作台、高速冷冻离心机、电泳仪、紫外分光光度计、凝胶成像系统、振荡器、移液器、吸头、Ep管、玻璃匀浆器、试管、 玻璃器皿洗净后置180?烘烤8h;不耐高温的器皿(如塑料制品)应用0.1% DEPC浸泡2h,70,80?烘烤干燥，120?高压20min，再70,80?烘烤干燥方可使用。 2.试剂: (1)细胞裂解液:异硫氰酸胍 4mol/L柠檬酸钠(pH7.0) 25mmol/L十二烷基肌氨酸钠 0.5%β-巯基乙醇 0.1mol/L 称取柠檬酸钠0.64g，十二烷基肌氨酸钠0.415g，吸取β-巯基乙醇0.7ml，用无Rnase的蒸馏水溶解，定容至50ml。然后取以上配制的溶液(CBS液)33ml，异硫氰酸胍 25g,混合，完全溶解后4?保存备用。 (2)10×凝胶缓冲液:吗啉代丙璜酸(MOPS)(pH7.0) 200mmol/L ,NaAc 100mmol/L,EDTA(pH 8.0) 10mmol/L 过滤除菌后避光保存。 (3)5×变性上样缓冲液: (水饱和的溴酚蓝 16μl 500mmol/L EDTA(pH 8.0) 80μl 甲醛(37%) 720μl 甘油 2ml 甲酰胺 3084μl 10×凝胶缓冲液 4ml 加无RNase水至10ml，4?可保存3个月。) influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there (4)TRIzol RNA抽提试剂 (5)2mol醋酸钠 (6)0.1% DEPC (7)平衡酚:氯仿:异戊醇(25:24:1) (8)平衡酚、氯仿 (9)无水乙醇、70%乙醇、异丙醇 (10)无RNase水 琼脂糖凝胶甲醛变性电泳检测 (1)制备凝胶(1.2%)。称1.2g琼脂糖，加72ml DEPC处理水，加热熔化。冷却至60?，在通风橱内加入10×凝胶缓冲液10ml，甲醛(37%)18ml，混匀后，倒胶。 2)制备样品。在离心管中，将RNA样品与5×变性上样缓冲液以4:1混匀。65?温育5,10min，迅速( 在冰上冷却5min，离心数秒。 (3)上样前凝胶须预电泳5min，随后将样品卷入上样孔。以5V/cm的电压电泳1.5,2h.。 (4)待溴酚蓝迁移至凝胶长度的2/3,4/5处结束电泳。将凝胶置于溴化乙啶溶液(0.5μg/ml，用0.1mol/L乙酸胺配制)中染色约30min。 (5)在凝胶成像系统观察并分析。 四、注意事项 1.RNA是极易降解的核酸分子。因此提取总RNA必须在无RNase 环境中，戴口罩、手套、使用无RNase污染的试剂、材料、容器。 2.所有溶液应加DEPC至0.05%,0.1%，室温处理过夜，然后高压处理或加热至70? 1 小时或60?过夜，以除去石油残留的DEPC。 3.所用的化学试剂应为新包装，称量时使用干烤处理的称量勺，所有操作均应在冰浴中进行，低温条件可减低RNA酶活性。 4.根据RNA样品的紫外吸收OD值，可计算出RNAd 浓度。 单链RNA [ssRNA]=40×(OD260-OD310)×稀释倍数 influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there 纯RNA OD260 / OD280比值通常在1.7,2.0。若比值低于1.7,说明有蛋白质等污染，应用酚/氯仿在抽提。 若比值低于2.0,表明有盐、胍、糖可用LiCL选择沉淀RNA以除去杂质。 influence and the weak. In recent years, diversification of sources of rural middle school teachers, teachers ' professional qualities and also more uneven, and many young teachers lack expertise, competition consciousness and ability in the workplace also lead directly to the loss of students, affecting the social reputation of the school, to the construction and management of rural middle school teachers sounds the alarm. Therefore, in a certain sense, attention to professional growth of young teachers in middle schools in villages and towns, focus on the future development of rural middle school. According to yingde green Tang town middle school Township middle school young teachers professional development research subject research programme, we subject group in objective real, and science actual, and development need of principles, design survey questionnaire, full volume total 69 problem, main content is divided into three a part: first part: basic information second part: teachers professional development status, and is divided into eight a part: a, and teachers expertise and skills II, and teachers workload and burden three, and education resources and textbook using four, and research and teaching situation five, and The six teaching reflective behaviors of teachers, teachers ' learning and training in seven, teachers on the professional identity of eight, the leisure part teacher professional development of teachers through questionnaires, questionnaires, record computer processes such as statistical analysis, cost of the sky survey report. Second, the purpose of the investigation, methods and objects (a) the purpose of the survey the purpose of the survey is that real, comprehensive, fully understand our school (if there