分子生物学实验手册 -高校教材

合集下载

2024年《分子生物学实验》课程教学大纲

《分子生物学试验》课程教学大纲课程编号： 05030适用专业：生物工程与生物技术试验学时数：36学时试验学分：1教材：主要参考书：《分子生物学试验指导》徐庆华等成栋学院立项教材 2024一、课程说明《分子生物学试验》以介绍分子生物学中的试验方法、试验手段和培育学生试验操作技能为其主要内容。

须要以分子生物学基本理论为基础，其教学目的是为了提高学生在分子生物学技术方面的动手实力，培育学生分析问题和解决问题的实力。

通过试验，要求学生能在原有的相关理论学问基础上较全面和深化理解分子生物学基本原理，驾驭基本的分子生物学试验方法和技巧，初步具备肯定的试验设计实力，以求为以后的学习和科研工作打下良好和扎实的基础。

本试验课在方法上力求经典，试验内容涉及了质粒DNA的提取及限制性内切酶酶切质粒分析；大肠杆菌感受态细胞的制备及质粒DNA转化大肠杆菌；应用多聚酶链式反应（PCR）体外基因扩增及产物鉴定；SDS－聚丙烯酰胺凝胶电泳测定蛋白质分子量等分子生物学的基本理论。

二、学时安排三、教学内容及教学基本要求试验一碱裂解法小量提取质粒DNA及琼脂糖凝胶电泳检测一、试验特点试验类型：综合试验类别：专业基础安排学时：6 每组人数：2二、试验目的1、驾驭碱裂解法小量提取质粒DNA和试剂盒提取质粒DNA的原理和方法。

2、驾驭琼脂糖凝胶的制备及外源DNA的检测原理。

3、了解质粒DNA的粗略定量方法。

三、试验内容提要1、将单菌落接种于3m1含相应抗生素的LB培育基中，37℃摇菌过夜；2、12,000rpm离心30sec，收集菌体；3、加200u1溶液I(含RNaseA 100ug/ml )，振荡悬浮菌体；4、加200ul新配制的溶液II，颠倒混匀；5、溶液澄清后马上加入预冷的200u1溶液III混匀后冰上放置5~10 min；6、4℃、12,000rpm离心15min，取上清，加0.7倍异丙醇混匀，室温静置5min；7、12,000rpm离心10min，70%乙醇洗涤沉淀，抽干后溶于适量水或TE，-20℃保存备用。

《分子生物学》实验指导(2015-2016)

《分子生物学》实验指导实验1 总DNA提取生物总DNA的提取是分子生物学实验的一个重要内容。

由于不同的生物材料细胞壁的结构和组成不同，而细胞壁结构的破坏是提取总DNA的关键步骤。

同时细胞内的物质也根据生物种类的不同而有差异，因此不同生物采用的提取方法也不同，一般要根据具体的情况来设计实验方法。

本实验介绍采用CTAB法提取植物总DNA的技术。

[实验目的]学习和掌握学习CTAB法提取植物总DNA的基本原理和实验技术。

学习和掌握紫外光吸收法鉴定DNA的纯度和浓度。

[实验原理]植物叶片经液氮研磨，可使细胞壁破裂，加入去污剂（如CTAB），可使核蛋白体解析，然后使蛋白和多糖杂质沉淀，DNA进入水相，再用酚、氯仿抽提纯化。

本实验采用CTAB法，其主要作用是破膜。

CTAB 是一种非离子去污剂，能溶解膜蛋白与脂肪，也可解聚核蛋白。

植物材料在CTAB的处理下，结合65℃水浴使细胞裂解、蛋白质变性、DNA 被释放出来。

CTAB与核酸形成复合物，此复合物在高盐（>0.7mM）浓度下可溶，并稳定存在，但在低盐浓度（0.1-0.5mM NaCl）下CTAB-核酸复合物就因溶解度降低而沉淀，而大部分的蛋白质及多糖等仍溶解于溶液中。

经过氯仿/ 异戊醇(24:1) 抽提去除蛋白质、多糖、色素等来纯化DNA，最后经异丙醇或乙醇等沉淀剂将DNA沉淀分离出来。

由于核酸、蛋白质、多糖在特定的紫外波长都有特征吸收。

核酸及其衍生物的紫外吸收高峰在260nm。

纯的DNA样品A260/280≈1.8，纯的RNA样品A260/280≈2.0，并且1μg/ml DNA 溶液A260=0.020。

[实验器材]1、高压灭菌锅2、冰箱3、恒温水浴锅4、高速冷冻离心机5、紫外分光光度计6、剪刀7、陶瓷研钵和杵子8、磨口锥形瓶（50ml）9、滴管10、细玻棒11、小烧杯（50ml）12、离心管（50ml）13、植物材料[实验试剂]1、3×CTAB buffer（pH8.0）100mM Tris25mM EDTA1.5M NaCl3% CTAB2% β-巯基乙醇2、TE缓冲液（pH8.0）10mmol/L Tris·HCl1mmol/L EDTA3、氯仿-异戊醇混合液（24：1，V/V）4、95％乙醇5、液氮[实验步骤]1、称取2g新鲜的植物叶片，用蒸馏水冲洗叶面，滤纸吸干水分。

分子生物学：基础实验技能培训实验操作手册

基础实验技能培训实验操作手册2014-08植物基因组DNA提取实验材料：植物样本，植物基因组DNA提取试剂盒，β-巯基乙醇，氯仿，无水乙醇实验仪器、耗材：移液器、电泳仪、离心机、研钵、研钵棒、剪刀、天平、液氮实验前准备：1．Buffer GP1在使用前请加入β-巯基乙醇，5 ml Buffer GP1加5 μl β-巯基乙醇。

加入β-巯基乙醇的Buffer GP1室温可保存1个月。

2．预热水浴锅65度，将加入配制好的巯基乙醇预热。

3．第一次使用前应按照试剂瓶标签的说明在Buffer GW1和Buffer GW2中加入无水乙醇。

4．称取植物样本约100 mg。

5．在研磨植物材料前，向研钵中倒入酒精，放入剪刀、钥匙高温消毒。

6．研钵、剪刀、钥匙冷却后，将液氮加入在研钵中以预冷研钵。

实验步骤：1. 取植物新鲜组织约100 mg，用剪刀将样本剪碎，放入研钵，加入液氮充分研磨至粉末状。

将粉末转移到离心管中。

注意：冻存材料直接研磨，绝对不能化冻。

而且粉末应在化冻前转移，否则内源性DNase 有可能降解基因组DNA。

2. 加入700 μl 65℃预热的Buffer GP1（Buffer GP1在使用前请加入β-巯基乙醇，5 ml BufferGP1加5 μl β-巯基乙醇），迅速颠倒混匀后，将离心管置于65℃水浴20分钟，水浴过程中颠倒离心管混匀样品数次。

3. 加入700 μl 氯仿，充分混匀，12,000 rpm（～13,400×g）离心5分钟。

小心将上层水相转入一新的离心管中，加入700 μl Buffer GP2，充分混匀。

4. 将上步所得溶液全部加入到已装入收集管（Collection Tube）的吸附柱（Spin Column DM）中。

若一次不能加完溶液，可分多次转入。

10,000 rpm（～11,500×g）离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

5. 向吸附柱中加入500 μl Buffer GW1（使用前检查是否已加入无水乙醇），10,000 rpm离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

赛默飞分子生物学手册

赛默飞分子生物学手册
分子生物学是一门关于生物分子结构、功能和相互作用的学科，它在现代生物学中起着至关重要的作用。

而赛默飞分子生物学手册则是一本被广泛使用的权威参考书，涵盖了各种生物分子研究方法和技术。

本手册的内容丰富多样，不仅包括了基础的概念和原理，还有最新的研究进展和实验操作指南。

在分子生物学领域，研究人员常常需要利用各种技术手段来探究生物分子的结构和功能。

赛默飞分子生物学手册提供了详细的实验步骤和操作方法，帮助研究人员快速准确地进行实验。

无论是DNA/RNA提取、PCR扩增、蛋白质分析还是基因表达等方面，本手册都能提供全面的指导，让科研工作更加高效和有序。

除了实验操作指南，赛默飞分子生物学手册还包含了大量的理论知识和研究进展。

从基本的生物分子结构和功能到前沿的基因编辑技术和细胞信号传导机制，本手册涵盖了分子生物学领域的各个方面。

研究人员可以通过阅读本手册，快速了解最新的研究动态，为自己的科研工作提供借鉴和启发。

此外，赛默飞分子生物学手册还提供了大量的案例分析和实验数据，帮助读者更好地理解和应用所学知识。

通过学习实际案例，研究人员可以更深入地了解分子生物学在不同领域中的应用和意义，为自己的研究方向找到更有力的论据和支持。

总的来说，赛默飞分子生物学手册是一本不可或缺的参考书，它为分子生物学领域的研究人员提供了全面的实验操作指南、理论知识和研究进展。

无论是初学者还是资深研究人员，都可以从中获益良多。

希望广大科研工作者能够善加利用这本手册，不断提升自己的研究水平，为生物科学的发展贡献自己的力量。

109分子生物学实验技术简册(pdf56)(1)

分子生物学常用实验技术第一章质粒DNA的分离、纯化和鉴定第二章 DNA酶切及凝胶电泳第三章大肠杆菌感受态细胞的制备和转化第四章 RNA的提取和cDNA合成第五章重组质粒的连接、转化及筛选第六章基因组DNA的提取第七章 RFLP和RAPD技术第八章聚合酶链式反应(PCR)扩增和扩增产物克隆第九章分子杂交技术第十章测序技术第一章质粒DNA的分离、纯化和鉴定第一节概述把一个有用的目的DNA片段通过重组DNA技术,送进受体细胞中去进行繁殖和表达的工具叫载体(Vector)。

细菌质粒是重组DNA技术中常用的载体。

质粒(Plasmid)是一种染色体外的稳定遗传因子,大小从1-200kb不等,为双链、闭环的DNA分子,并以超螺旋状态存在于宿主细胞中。

质粒主要发现于细菌、放线菌和真菌细胞中，它具有自主复制和转录能力,能在子代细胞中保持恒定的拷贝数,并表达所携带的遗传信息。

质粒的复制和转录要依赖于宿主细胞编码的某些酶和蛋白质，如离开宿主细胞则不能存活,而宿主即使没有它们也可以正常存活。

质粒的存在使宿主具有一些额外的特性,如对抗生素的抗性等。

F质粒(又称F因子或性质粒)、R质粒(抗药性因子)和Col质粒(产大肠杆菌素因子)等都是常见的天然质粒。

质粒在细胞内的复制一般有两种类型:紧密控制型(Stringent control)和松驰控制型(Relaxed control)。

前者只在细胞周期的一定阶段进行复制,当染色体不复制时,它也不能复制,通常每个细胞内只含有1个或几个质粒分子,如F因子。

后者的质粒在整个细胞周期中随时可以复制,在每个细胞中有许多拷贝,一般在20个以上,如Col E1质粒。

在使用蛋白质合成抑制剂-氯霉素时,细胞内蛋白质合成、染色体DNA复制和细胞分裂均受到抑制,紧密型质粒复制停止,而松驰型质粒继续复制,质粒拷贝数可由原来20多个扩增至1000-3000个,此时质粒DNA占总DNA的含量可由原来的2%增加至40-50%。

分子生物学操作手册

分子生物学操作手册一、引言分子生物学是研究生物体分子结构、功能和相互作用的一门学科。

操作手册旨在提供分子生物学实验的详细步骤，并帮助读者准确、高效地进行实验操作。

本文将介绍PCR扩增、DNA电泳、亚克隆和蛋白质表达等实验步骤，并提供一些常用操作的技巧和注意事项。

二、PCR扩增PCR扩增是分子生物学实验中常用的技术，用于扩增特定区域的DNA片段。

以下是PCR扩增的基本步骤：1. 准备PCR反应体系：根据实验需要，配置PCR反应液，包括DNA模板、引物、酶、缓冲液和dNTPs等。

2. 设定PCR程序：根据模板序列的长度和引物的特性，设定合适的PCR程序，包括变性、退火和延伸等步骤。

3. PCR反应：将PCR反应体系置于热循环仪中，按照设定的程序进行PCR反应。

4. 结果分析：利用DNA电泳等技术，分析PCR反应产物的大小和纯度。

三、DNA电泳DNA电泳是一种常用的DNA分析技术，用于确定DNA片段的大小和纯度。

以下是DNA电泳的基本步骤：1. 制备琼脂糖凝胶：根据需要，配制适当浓度的琼脂糖凝胶，并倒入凝胶槽中，形成凝胶床。

2. 加载样品：将PCR扩增产物或其他DNA样品与DNA标记物混合，加入琼脂糖凝胶孔中。

3. 进行电泳：将凝胶槽放入电泳仪中，进行电泳操作，根据需要设置适当的电压和时间。

4. 结果分析：利用紫外线透射仪观察凝胶上DNA迁移的情况，测量DNA片段的大小。

四、亚克隆亚克隆是将DNA片段插入到载体DNA中的过程，通常用于构建重组DNA或进行基因克隆。

以下是亚克隆的基本步骤：1. 准备载体和DNA片段：准备含有目标基因的DNA片段和经酶切的载体DNA。

2. 消化与连接：将DNA片段与载体DNA进行连接反应，通常使用DNA连接酶。

3. 转化：将亚克隆反应产物转化到适当的宿主细胞中，如大肠杆菌。

4. 筛选与鉴定：通过筛选培养基和PCR等技术，鉴定含有目标基因的克隆。

五、蛋白质表达蛋白质表达是将目标蛋白质在细胞内大量合成的过程，利用该技术可以生产重组蛋白以供研究和应用。

分子生物学实验手册

所有资源请在下载后 24 小时内删除。

如果您觉得满意，请购买正版，以便更好支持您所喜欢的软件或书籍！☆☆☆☆☆生物秀[]☆☆☆☆☆中国生物科学论坛[/bbs/]☆☆☆☆☆生物秀下载频道[/Soft/]生物秀——倾力打造最大最专业的生物资源下载平台！■■■ 选择生物秀，我秀我精彩！！■■■欢迎到生物秀论坛（中国生物科学论坛）的相关资源、软件版块参与讨论，共享您的资源，获取更多资源或帮助。

2000/01 Cardiff School of BiosciencesMolecular Biology IBiochemistryLevel II:PracticalManualDr Peter KilleGENE CLONING PRACTICALThe purpose of this practical is to introduce you to the basic techniques of gene cloning. The techniques that you will use include:The preparation of plasmid DNADigestion of DNA with restriction endonucleasesAnalysis of DNA fragments by electrophoresisLigation of DNA fragments to produce recombinant moleculesTransformation of bacterial cellsSelection of clones by insertional inactivation of a geneSelection of clones by expressed phenotypesAmplification of specific DNA fragments by the polymerase chain reaction (PCR)Induction of gene expressionEnzyme activity measurementParticular attention should be paid to the following points:[1] The manual should be used as a guide to assist you with the experiments rather than asa complete recipe book.[2] There are certain safety requirements (in addition to those appropriate to normallaboratory practice) that must be adhered to (see next page). It is a requirement that you read the section on safety before commencing any work.[3] These experiments will work well but only if the advice on page 4 is followed.TIMETABLEPracticals will be held in lab. 101W every Tuesday. Sessions will start promptly at 10.00 am and will continue through until 18.00. If you cannot attend any part of the practical please inform a demonstrator or the lecturer in charge.Week 1[1] Preparation of plasmid DNA[2] Digestion of DNA with restriction endonucleases[3] Analysis of DNA fragments by electrophoresis[4] Ligation of DNA fragments to produce recombinant moleculesWeek 2[5] Transformation of bacterial cells[6] Plating out of bacteria on selective mediaWeek 3* Monday before practical (approx. 1 hour)[7] Plating out of bacteria for insertional and phenotypic screening* Tuesday[8] Selection of clones by insertional inactivation of a gene[9] Selection of clones by expressed phenotype[11] Preparation of plasmid DNA from recombinant plasmids[10] Identification of specific gene by polymerase chain reaction (PCR) and restrictionmapping.Week 4* Monday before practical (approx. 1 hour)[11] Inoculation of cultures for induced gene expression* Tuesday[12] Analysis of induced gene expression by enzyme activity measurements[13] Analysis of PCR and restriction fragments plasmids prepared in [10].SAFETYGeneral PointsAll experiments involving recombinant DNA work must be approved by a local safety committee who are responsible for quantifying the biological risk and designating the level of containment that is required for the work to proceed. The committee has a legal obligation to submit annual reports on all experiments carried out in College to the Health and Safety Executive. This experiment has been approved subject to the work being carried out in accordance with 'good microbiological practice'.Specific PointsIn addition to the safety procedures that are applicable to any laboratory work the following points should be noted.[1] Experiments must be carried out using good microbiological practice. This meansthat the normal safety precautions applicable to the handling of microorganisms apply to the experiments described in this manual.[2] Compounds that interact with DNA, e.g. ethidium bromide, are mutagens andcarcinogens. Do not allow these compounds to come into contact with yourself orother people. Wear disposable gloves at all times.[3] The transilluminator is a powerful source of ultra violet radiation and will causeserious damage to unprotected skin and eyes. Always ensure that there is a uv-opaque material between you and the light source, i.e. wear gloves and use suitable eye/faceprotection.[4] Phenol-containing solutions can cause severe burns. Make sure that you are wearinggloves when handling these solutions.[5] If you have recently been prescribed medicines by your doctor, especially antibiotics,anti-inflammatory drugs or immunosuppresants, please inform Dr P Kille beforestarting the practical.PRACTICAL CONSIDERATIONSEssentially, the basic techniques of gene cloning are straightforward providing that certain protocols are followed. These practical points are important if you are to obtain successful results.[1] Keep solutions on ice at all times. Even short exposure of certain components, e.g.some restriction endonucleases, to room temperature will cause significantinactivation.[2] Nucleases are ubiquitous and some are extremely stable enzymes. It is vital that theseenzymes are not allowed to come into contact with your apparatus or solutions.Always wear disposable gloves - the skin is an excellent source of nucleases and there is enough enzyme in a fingerprint to ruin your experiments. Also, most of yoursolutions and apparatus have been autoclaved to destroy nucleases so avoidunnecessary handling.[3] The key to many of the techniques described in this manual is the ability to pipettesmall volumes carefully and accurately. It is essential when pipetting small volumes to ensure that the tip of the pipette is touching the surface of the container beforeexpelling the solution. Always use clean pipette tips to avoid cross contamination.Pulse spin microfuge tubes to ensure that all of the added components of reactionmixtures are mixed at the bottom of the tube.[4] The transilluminator should be used for the minimum time possible as prolongedexposure of DNA samples to uv radiation causes random strand breakage - also thefilter has a finite life. Please avoid scratching the surface of the transilluminator asthis will destroy the effectiveness of the filter - a new transilluminator costs £1000.A general point about recombinant DNA work is that reagents are extremely expensive. Please use reagents/enzymes as sparingly as possible. In general, expensive components such as enzymes will have been pre-dispensed into tubes. The other components of the reactionmixture should be added to these tubes (see protocols for further details).BACKGROUND TO THE EXPERIMENTSAlginates are polysaccharides comprised of (1-4)-linked β-D-mannuronate andα-L-guluronate. This polysaccharide is produced by marine macroalgae and certain bacteria, and the algal alginate is widely used by the food and pharmaceutical industries (approx. 22000 tonnes/annum).Certain bacteria produce enzymes (alginate lyases or alginases) which degrade alginates. A gene encoding an alginate lyase has been cloned from the bacterium Klebsiella pneumoniae into a cosmid vector to produce the construct pSP1. In this practical the bacterium E. coli strain LE392 will be used as a source of plasmid pSP1. This plasmid contains the aly gene which encodes the alginate lyase (Fig. 1).Objectives of the Practical[1] Both the plasmid vector (pHG327) and the 'foreign DNA' (pSP1) will be isolated andpurified from cultures of E. coli.[2] Both pHG327 (Fig. 2) and pSP1 will be cut with an appropriate restrictionendonuclease (Hin dIII).[3] Fragments of pSP1 will be added to the cut pHG327 and recombinant molecules willbe allowed to form. The recombinant molecules will be treated with DNA ligase to reform the phosphodiester linkages.[4] Bacterial colonies will be screened on MacConkey agar to detect transformants.[5] Recombinants containing the aly gene will be isolated by detecting expression of thealginate lyase enzyme.[6] DNA will be isolated from recombinants, cut with restriction endonucleases and the fragments analysed on agarose gel electrophoresis. This will allow the aly gene to be mapped onto pSP1.[7] The polymerase chain reaction (PCR) will be used to identify the HindIII fragment of the pSP1 that contains the aly gene from crude bacterial extracts.[8] The expression of alginate lyase by recombinants will be quantified by measurement of specific enzyme activities.HindIIIHindIIIFig. 1 Map of pSP1(Positions of restriction sites are approximate)(Low copy number) (High copy number)Fig. 2 Map of pHG327BACTERIA, ENZYMES AND ANTIBIOTICSBacterial strainsYou will use one or more of these strains of Escherichia coli which have the following genotypes:Escherichia coli JM109 ∆(lacpro AB),end A1, rec A1, thi, sbc B15, hsd R4, sup E44, rel A1(F', tra D36, pro AB+, lac I q Z∆M15)Escherichia coli JM107 ∆(lacpro AB), end A1, gyr A96, thi-1, hsd R17, sup E44, rel A1, (F',tra D36, pro AB+, lac I q Z∆M15) (pHG327, Ap r)Escherichia coli LE392 hsdR514 (rk-,mk+), supE44, supF58, lacY1 (pSP1, Km r)Some genetic markers in frequently used E. coli strains (Data from Promega)Symbol Description Effectend A Endonuclease mutation Improves quality of plasmid DNA isolationsF' Host contains F' episome Provides essential functions to host cellgyr DNA gyrase mutation Confers resistance to nalidixic acidhsd R Restriction system mutations Allows cloning without cleavage of plasmid DNAlac repressor protein Inhibits transcription from lac promoterthelac I q Overproduceslac Y Galactose permease mutation Blocks lactose utilizationlac Z β-D-Galactosidase mutation Unable to produce β-D-galactosidase. Strainssuitable for use with lac Z-containing vectorspro AB Mutations in proline metabolism Requires proline for growth in minimal medium rel A Mutation eliminating stringent factor RNA synthesis in the absence of protein synthesis sbcB15Exonuclease I mutation Allows general recombination in rec BC mutants sup E Supressor mutation Suppresses amber (UAG) mutationssup F Supressor mutation Suppresses amber (UAG) mutationstra D Transfer factor mutation Prevents transfer of F' episomerec A1 Mutation in recombinantion Prevents recombination of introduced DNA withhost DNA.Restriction enzymesEnzyme Microorganism SpecificityBam HI Bacillus amyloliquifaciens H G\GATCCEco RI Escherichia coli RY 13 G\AATTCHin dIII Haemophilus influenzae Rd A\AGCTT164 CTGCA\GstuartiiPst I ProvidenciaXho I Xanthomonas holcicola C\TCGAGAntibioticsAp AmpicillinKm KanamycinAp r Ampicillin-resistantphenotypephenotypeAp s Ampicillin-sensitiveMINI-PREPARATION OF DNAExtraction of Plasmid DNA from bacterial cells.Materials/Solutions Required5ml culture of E. coli JM 107 containing pHG3275ml culture of E. coli LE392 containing pSP1Collection Tubes.Sterile water warmed to 70°CSV Mini prep Kit (The SV Miniprep Kit is manufactured by Promega)Cell resuspension solution (50mM-Tris, pH 7.5; 10mM-EDTA; 100µg/ml RNase A) Cell lysis solution (0.2M-NaOH, 1% SDS)Alkaline Protease solutionNeutralisation solution (4.09M Guanidine hydrochloride, 0.759M-Potassium acetate,2.12M Glacial acetic acid)SV spin columnsSV Column wash solution (162.8mM Potassium Acetate, 27.1mM Tris-HCl, pH7.5 -EDTA - Add 1.75 volumes of 95% ethanol before use i.e. 35mls of ethanol to 20mls Wash concentrate).MethodYou will be provided with bacterial cultures that have been grown overnight at 37°C in medium containing appropriate antibiotics.[1] Pellet bacterial cells by centrifuging 5ml of each bacterial culture for 10mins at 3K ina bench top centrifuge.[2] Remove the clear supernatant. Add 250µl of Cell Resuspension Solution andresuspend the cell pellet by brief vortexing.[3] Transfer each cell suspension to separate sterile eppendofs.[4] To each add 250µl of Cell Lysis Solution and mix by inverting the tube four times (donot vortex). Incubate for 1-5 minutes at room temperature until solution clears.10µl of Alkaline Protease solution and mix by inverting the tube 4 times. [5] AddIncubate for a further 5 minutes at room temperature. Do not incubate for longerthan 5 minutes350µl of Neutralisation Solution and immediately mix by inverting the tube [6] Addseveral times.[7] Spin in the microcentrifuge for 10 min.A. Purification with Centrifugation.[8A] Transfer the cleared lysate to a Spin Column by decanting. Avoid disturbing of transferring any of the white precipitate with the supernatant. Insert the Spin Column into a collection tube.[9A] Spin in the microcentrifuge for 1 min. Remove the spin column from the tube and discard the flowthrough from the Collection tube. Reinsert the Spin Column into the Collection Tube.[10B] Add 750µl of Column Wash solution (previously diluted with ethanol) to the Spin Column.[11A] Spin in the microcentrifuge for 1 min. Remove the spin column from the tube and discard the flowthrough from the Collection tube. Reinsert the Spin Column into the Collection Tube.[12A] Add 250µl of Column Wash solution (previously diluted with ethanol) to the Spin Column.[13A] Spin in the microcentrifuge for 2 min.[14A] Transfer Spin Column to a new sterile eppendoff.B. Purification with Vacuum.[8B] Insert the Spin Column into vacuum manifold. Transfer the cleared lysate to a Spin Column by decanting. Avoid disturbing of transferring any of the white precipitatewith the supernatant.[9B] Apply vacuum to pull liquid through the column. When all liquid has been pulled through column, release vacuum.[10B] Add 750µl of Column Wash solution (previously diluted with ethanol) to the Spin Column.[11B] Apply vacuum to pull liquid through the column. When all liquid has been pulled through column, release vacuum.[12B] Add 250µl of Column Wash solution (previously diluted with ethanol) to the Spin Column.[13B] Apply vacuum to pull liquid through the column. When all liquid has been pulled through column, release vacuum. Transfer to Collection tube and spin in themicrocentrifuge for 2 min to remove any residual wash solution.[14B] Transfer Spin Column to a new sterile eppendoff (with the lid cut off).DNA Elution[15] Elute the plasmid DNA by adding 100µl of nuclease-free water (at 65-70°C) to theMinicolumn.[17] Spin the microfuge tube and column for 1 min in a microcentrifuge.[18] Remove and discard the Minicolumn but keep the liquid. The plasmid DNA, which iscontained in the remaining liquid should be transferred with a pipette to a freshmicrofuge tube (with a lid) and may be stored at 4°C or -20°C until required.The rationale behind the various steps in the experimental protocol is as follows:Step 3 Cell resuspension solution contains EDTA which makes the bacterial outer membrane permeable and RNase which degrades RNA.Step 4 The NaOH denatures the proteins and disrupts the chromosome. The detergent, SDS, lyses the bacteria allowing the plasmids to leak out of thecells. Alkaline Protease breaks down bacterial proteases.Step 5 The guanidine hydrochloride and potassium acetate forms a precipitate with the proteins and chromosomal DNA while the plasmid remains in solution.The acetic acid neutralises the alkali used in the previous step.Step 6-7 The plasmid (in solution) is separated from the precipitated proteins,chromosome and cell debris.Step 8- Under high salt conditions Nucleic acids binds (plasmid DNA) to Spin Column.Step 9-13 Contaminating proteins and salts are washed away. The washing solution contains salt ethanol and a small amount of salt so that mono-nucleotides areremoved but plasmid DNA remains bound.Steps 14 Remaining wash solution is removed.Steps 15-18 Water at 65°C causes the DNA to be released from the resin. The plasmid DNA solution is then collected in a fresh eppendoff when the column is spunin the microcentrifuge.RESTRICTION ENDONUCLEASE DIGESTION OF DNApSP1 is digested into a number of fragments using the restriction enzyme Hind III. The expression vector pHG 327 is also cut with Hind III, in a single location, causing it to linearise. The aim is to ligate the pSP1 fragments into the cut pHG 327. However, when the ligation is performed there is also the possiblity that pHG 327 will reform without incorperating any fragments of pSP1. To prevent this we treat the linearised pHG 327 with a Shimp dephosphorylase enzyme which removes the 5’ phosphate groups making it only possible for the pHG 327 to reform if a fragment from pSP 1 is incorrperated.Materials/Solutions Required10X Digest bufferHin dIII enzymeDNA sampleSterile waterMethod[1] Decide a suitable volume of each plasmid DNA and the final volume of digestion.digest Preparativedigest AnalyticalpHG327 pSP1 DNA volume 10µl 15µl 25µl Final volume 20µl 30µl 50µl [2] You will be provided with tubes containing 2µl of restriction enzyme. To each tubeadd 1/10 final volume of 10X Buffer, sample DNA and sufficient sterile water to give the appropriate final volume.[3] Flick the bottom of the tubes to mix the contents (remember that it is easy to denaturethe enzyme) and then microfuge for 2-3 seconds so that all of the contents are at thebottom of the tube.[4] Incubate the tubes at 37°C for 2 hours (see “DEPHOSPHOYLATION OF VECTORDNA” for instructions of what to do with pHG327 digestion) and then transfer thetubes to a 65°C waterbath for 15 min to inactivate the endonuclease anddephosphoylation enzyme.[5] Use a portion (e.g. 7-8µl) of the digest for analysis by gel electrophoresis and store theremainder for use in the ligation reaction.DEPHOSPHOYLATION OF VECTOR DNAMaterials/Solutions RequiredShrimp PhosphataseMethodpHG327 digestion only.[4a] After the pHG327 Vector DNA has been incubating with Hin dIII for 1 hour remove it from the water bath and add 1µl of Shrimp Phosphatase. Flick the bottom of the tubes to mix the contents and then microfuge for 2-3 seconds so that all of the contents are at the bottom of the tube.[4b] Place the digestion mixture in the water bath to incubate at 37°C for an additional1 hour and then continue as for normal digestion’s at step [5].FORMATION OF RECOMBINANT DNA MOLECULESThe digested pSP1 fragments are ligated into the linearised dephosphorylated pHG 327. Before the ligation is performed the digested DNA will be purified with chloroform/isoamyl alcohol and concentrated by precipitation with ethanol.Materials/Solutions RequiredHin dIII digested DNA samplesSterile waterChloroform/iso amyl alcoholTE buffer3M sodium acetate100% ethanol (at -20°C)10X Ligation bufferT4 DNA ligaseMethod[1] Add the following components in a microfuge tube placed on ice:Dephosphorylated Hin dIII digested pHG327 (vector) DNA 20µlHin dIII digested pSP1 (foreign) DNA 40µlSterile diH2O 40µlTotal 100µl 100µl of chloroform/iso amyl alcohol to the tube, vortex and microfuge for [2] Add2 min.[3] Transfer the aqueous (upper) phase to a fresh tube. It is essential that none of thechloroform phase is transferred.10µl of 3M sodium acetate and 250µl of cold ethanol to the tube. Leave in an [4] Addethanol/dry ice bath (or at -70°C) for about 30 min.[5] Microfuge for 15 min, carefully pour off the supernatant and dry the pellet (may notbe visible). Be careful not to lose the pellet.[6] Resuspend the pellet in 14µl of sterile water[7] You will be provided with a tube containing 2µl of T4 DNA ligase. To this, add 2µlof 10X ligation buffer, the resuspended pellet (14µl) and gently mix.[8] Briefly microfuge to bring contents to the bottom of the tube and incubate at 16°Covernight.TRANSFORMATION OF HOST BACTERIA WITH RECOMBINANT DNAThe recombinant DNA molecules form during the ligation are inserted into E. coli cells and the bacteria containing the plasmids are selected by their resistance to Ampicillin.Materials/Solutions Required50µl competent E. coli DH5α on ice.1 ml Sterile S.O.C. Media.Ligated DNA sampl.eLB Agar capsules.100 mg/ml Amp.Method(a) Preparation of Agar plates[1] Put 8 capsules of LB Agar into the provided 500 ml Duran Bottle and add 200 mls ofSterile water. Screw cap on LEAVING IT LOOSE and cover top with tin foil. Adda strip of autoclave tape and place into autoclave for 20 minutes at 120 p.s.i..[2] When autoclave cycle is finished USE TERMAL RESISTANT GLOVES toremove molten agar from autoclave. Tighten lid and swirl to insure capsules are fully dissolved and place in 55°C water bath.[3] Check molten L Agar is at 50°C remove and pour one agar plate ~20 mls (make surethis plate is labled “LB-AGAR NO ANTIBIOTIC”) then to the remainder add Amp toa final concentration of 100 µg/ml.[4] Mix by swirling and place back into 50°C waterbath for 2-3 minutes until majority ofbubbles disappear.[5] Carefully pour media evenly into 6 sterile petri dishes using good aseptic technique.Any excess LB-agar should be washed down the sink immeadiately with lots of warm water.[6] Wait for plates to solidify (~30 minutes) and dry by opening slightly and placingupside down in the 37°C incubator.[7] Once dry close plate and leave on bench ready for transformation.E. coliof(b) Transformation[8] Add the DNA sample (maximum volume 5µl) to 50µl of competent cells and gentlytap the tube to mix.[9] Incubate the tubes on ice for 30 minutes. Then at 37°C for 20 seconds and thenreplace them on ice for an additional 2 minutes.[10] Add 0.95ml of SOC media and incubate at 37°C for 1 hour.[11] *Label the plates before adding bacteria.[12] Spread 150 µl portions of the transformed bacteria onto 6 of your LB Amp plates(prepared in Section a) and the last 100 µl portion on the LB plate containing noantibiotic. *Label remaining LB Amp plate and store in fridge for use in following week.[13] Incubate upright until the agar surface is dry before inverting the plates and leavingovernight at 37°C.SCREENING CLONESRecombinant clones (i.e. those colonies which contain pHG 327 with a pSP1 insert) will be identified by insertional activation (described in rational section). The recombinant clones identified may contain any of the five fragments produced by digestion of pSP1 with Hind III. In order to determine which of these recombinant clones contains the aly gene (encoding alginate lyase), the ability of the colonies to degrade alginate is assessed.Materials/Solutions RequiredMacConkey plate (contain 100µl Ap/ml)Alginate plate (contain 100µl Ap/ml)Sterile applicator sticksCulture dish containing 24 wells each holding 2mls of LBamp media.Method : Monday Evening[1] Examine the plates that you have prepared previously. The LB plate with noantibiotic should show lawn growth whilst you should be able to identify singlecolonies on the LB Amp plates. Only bacteria containing pHG327 or pHG327 plusinsert DNA should grow on LB Amp plates.[2] On back of MacConkey plate and Alginate plate draw a 6 x 4 grid and label eachsquares A1-A6, B1-B6, C1-C6 and D1-D6. Wells in culture dishes are alreadylabelled in the same manner, check dish orientation.[3] Pick selected individual colony from LB Amp plates with Sterile applicator stick.Place gently onto square A1 of alginate plate, then A1 of MacConkey plate and finally into well A1 of culture dish. Repeat this procedure with new collonies until allsquares all filled (total 24 collonies).[4] Incubate the plates overnight at 37°C.Tuesday Morning[5] Count the number of red colonies which are Lac+ bacteria, and the white colonieswhich are Lac- on the MacConkey agar plate. Note location of white collonies.[6] Overlay the alginate plates with 10%(w/v) cetyl pyridinium chloride (CPC) solutionand leave for 5-10 min. Undegraded alginate will appear opaque white and colonies producing alginate lyase will be surrounded by a clear zone. Placing the plates on ablack background will help you to visualise the clearing zones. CPC is bacteriocidal, therefore you will need to utilise the corresponding colonies on the MacConkey agar plates for further study. Note location of colonies that give clearing zones.[7] Identify i) Collony that is white on MacConkey agar plate and gives clearing zone. ii)Collony that is red with no clearing zone. Take spare LB AMP plate prepared inprevious week, divide in half and using sterile applicator sticks streak out these twocollonies. Give plate to demonstrators at end of session.[8] Locate the wells in culture dish that contain the liquid replicate cultures of the thosecollonies identified in [7]. Prepare bacterial DNA from these cultures and anaylse by performing restriction digests (analytical, as for pHG 327) and PCR.The rationale behind each step of the experimental protocol is as follows:Step 1 Lawn growth on L agar indicates that the competent cells were viable. No growth on MacConkey agar (containing ampicillin) indicates that the host cells were sensitive to ampicillin and cannot grow on this medium.Step 2 pHG327 confers antibiotic resistance on the host cells, therefore the transformants should be able to grow on medium containing ampicillin.Step 3 The vector pHG327 contains the lacZ gene encoding β-galactosidase, a gene which has been partially deleted in the particular host cells that we have used. Therefore, it is only the bacteria containing intact pHG327 that are able to metabolise lactose(present in MacConkey agar) to produce acid which turns the indicator phenol red(also present in MacConkey agar) from colourless to red. Those colonies that remain white should contain genuine recombinant DNA molecules. The cloning site inpHG327 is contained within the lacZ gene. Therefore, if a piece of foreign DNA has been inserted into this site the lacZ gene will be disrupted and no β-galactosidase will be produced. This phenomenon is called 'insertional inactivation' of a marker gene.Step 4 Duplicate plates are required of each colony because the alginate lyase detection procedure (step 6) kills the bacterial cells.Step 6 CPC is a cationic detergent which binds to high molecular weight alginates to form an opaque white precipitate. Alginate lyase reduces the molecular weight of the alginate sufficiently to prevent a significant precipitate from forming, hence the appearance of 'clearing zones' around alginate lyase producing colonies.AGAROSE GEL ELECTROPHORESISDNA may be fractionated according to molecular size by electrophoresis through agarose gels. This method will be used to analyse intact plasmids and also DNA fragments produced by restriction endonuclease digestion.Materials/Solutions RequiredAgarose (0.7%) in 1 X TBEElectrophoresis buffer - 5 X TBE (0.445M Tris, 0.445M boric acid and 0.01M EDTA, pH8.0) Electrophoresis apparatusEthidium bromide solution (2µg/ml)Masking tapeMethod[1] Seal the ends of the gel holder with tape.[2] Place comb in the correct orientation across the end of the gel holder.[3] Melt agarose in the autoclave.[4] Check that agarose is no hotter than 55°C and pour into the holder taking care to avoidair bubbles. Allow to set (approx. 20 min). Do this with the gel holder on the bench - not in the electrophoresis apparatus.。

高中生物实验指南：分子生物学实验操作手册

高中生物实验指南：分子生物学实验操作手册1. 简介分子生物学是研究生物体内分子结构、组成、功能以及相互作用的科学。

在高中生物学课程中，通过进行一系列分子生物学实验，可以加深对基因、DNA、RNA等内容的理解，并培养学生动手实践和科学探究的能力。

本操作手册将介绍一些适合高中副标准课程的基本分子生物学实验，帮助教师和学生更好地开展这方面的实验教学。

2. 实验一：提取DNA2.1 实验目的了解DNA的结构和提取过程，并掌握提取DNA的基本方法。

2.2 材料与仪器•成熟香蕉或其他植物材料•盐水溶液（含氯化钠）•蛋白酶（如葡萄胺酸酶）•洗涤剂（如洗衣粉）•咖啡滤纸或滤纸漏斗•烧杯•酒精1.将香蕉剥皮后放入砧板上，用刀将香蕉捣碎成泥状。

2.在烧杯中加入适量的盐水溶液，并将捣碎的香蕉放入其中搅拌均匀。

3.加入少量蛋白酶和洗涤剂，再次搅拌均匀，让细胞破裂释放DNA。

4.将混合物过滤到另一个烧杯中，通过滤纸除去大颗粒的残渣。

5.将过滤后的溶液倒入试管中，并缓慢地倾斜试管，在溶液与酒精接触的界面处，会出现白色粘稠物质，即提取到的DNA。

2.4 实验结果及分析实验中提取到的DNA呈现为白色细丝状物质。

通过这个实验可以观察到DNA 在水相和酒精相之间的界面上凝聚堆积。

说明了DNA在酒精中不溶性，从而便于提取。

3. 实验二：PCR扩增3.1 实验目的了解聚合酶链式反应（PCR）的原理和基本步骤，并能够进行PCR扩增实验。

3.2 材料与仪器•PCR试剂盒（含模板DNA、引物、聚合酶等）•热循环仪•PCR试管或反应管1.准备PCR反应体系，按照试剂盒说明书的要求加入模板DNA、引物和聚合酶等。

2.将装有反应溶液的PCR管放入热循环仪中。

3.设置PCR的温度程序，包括变性、退火和延伸阶段。

4.启动热循环仪开始PCR扩增反应。

5.完成PCR后，可以通过凝胶电泳等方法检测扩增产物。

3.4 实验结果及分析通过PCR扩增，可以在较短的时间内大量复制并放大目标基因片段。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

实验二质粒 DNA 的提取
实验目的
本实验通过质粒快速提取试剂盒的试剂对大肠杆菌中的重组质粒 pET-22b 的抽提，掌握质粒 DNA 的小量制备方法，了解碱裂解法制备质粒 DNA 的基本原理。
实验原理
载体是基因工程所必需的工具，它用于将目的基因运载到宿主细胞内进行克隆与表达。常用的载体有质粒、λ噬菌体、M13 噬菌体、逆转录病毒 DNA 和昆虫病毒 DNA 等，而质粒是最常用的载体。
南京工业大学制药与生命科学学院
《分子生物学实验》教学大纲
英文名称： Molecular biology
学分：1
学时：32
教学对象：生物工程专业、制药工程专业、药剂学专业、食品学专业、生物技术
专业
教学目的：在开设的理论课程的基础上，结合实验课程的教学，使学生能较全面系统的掌握分子生物学的基本操作，拓宽专业知识面，加深对专业知识的理解，强化动手能力。。
电泳图
凝胶成像系统
实验四细菌基因组 DNA 的提取
实验目的
本实验通过利用小量细菌基因组 DNA 抽提试剂盒对大肠杆菌基因组 DNA 的抽提，掌握细菌基因组 DNA 的小量制备方法。
实验原理
本实验使用溶菌酶破壁及蛋白酶 K 消化以确保基因组 DNA 得率。不使用苯酚/氯仿等有机试剂，无须传统的乙醇沉淀、洗涤、干燥、溶解过程，简化了操作。
实验原理
琼脂糖凝胶电泳是分子克隆的核心技术之一，可用于分离、鉴定和纯化 DNA 片段。胶回收是指目的 DNA 片段经由琼脂糖凝胶电泳与其它片段分离，再从琼脂糖凝胶中将目的 DNA 片段抽提纯化出来的过程。
实验步骤
操作流程见右图 1 目的 DNA 电泳。 2 切出含有目的 DNA 的凝胶。 3 称量胶块重量，计算胶块体积。 4 向胶块中加入胶块融化液。 5 75℃加热融化胶块。 6 将溶液转移至 Spin Column 中离心 1 分钟，弃滤液。 7 用 Rinse A，Rinse B 冲洗 Spin Column。 8 在 Spin Column 膜的中央处加入 25 μl 的灭菌蒸馏水或 Elution Buffer12,000 rpm 离心 1 分钟洗脱 DNA。 9 取胶回收目的基因片段洗脱液 1 μl 加上 10×Loading Buffer 1μ l 混匀。
重点:
掌握质粒 DNA 的小量制备方法，了解碱裂解法制备质粒 DNA 的原理。进一步
了解限制性内切酶的特性，掌握 DNA 的酶切技术。
难点: 掌握质粒 DNA 的小量制备方法及 DNA 的酶切技术。
实验二. 细菌基因组 DNA 的提取
（4 学时）
基本要求:
了解细菌基因组 DNA 的提取的目的，原理，掌握细菌基因组 DNA 的提取的实
离蛋白质的基本步骤及实验结果的判定难点:
聚丙烯酰氨凝胶电泳的实验步骤及实验结果的判定
实验一氯化钙法大肠杆菌感受态细胞的制备及转化
实验目的
掌握重组 DNA 质粒转化至大肠杆菌宿主的操作技术。
实验原理
带有外源 DNA 的重组质粒，在体外构建后，导入宿主细胞，随着细胞的大量复制、繁殖，才能够有机会获得纯的重组质粒 DNA,,该过程称之为转化过程。受体细胞经过一些特殊方法（如：CaCl2，RuCl 等化学试剂法）的处理后，细胞膜的通透性发生变化，能容许外源 DNA 的载体分子通过。
实验步骤
大肠杆菌形态图
1 感受态细胞的制备： 1) 挑菌 37℃培养。 2) 将过夜培养的细菌转接培养 2-3 小时。 3) 将菌液置冰上 10 分钟，4℃离心 8000rpm 30 秒。 4) 用氯化钙溶液悬浮菌体，冰浴，离心弃去上清。 5) 氯化钙溶液悬浮菌体。 2 转化： 1) 取出感受态细胞,试剂 A，无菌双蒸水和待转化的质粒 DNA，均置冰盒内。 2) 取离心管管一支，将质粒、试剂 A、无菌水，感受态细胞混匀冰浴 20 分钟。 3) 在管内加入 400ul LB 液体培养基，振荡培养 10-40 分钟。 4) 涂布培养。
实验五质粒 DNA 限制性内切酶实验
实验目的
通过 DNA 限制性内切酶实验掌握内切酶技术。
实验原理
不同酶切反应都需要不同的酶切缓冲液。多数生物技术公司的产品目录中均有关于何种限制性内切酶适合何种缓冲液的资料可供查阅。绝大多数酶的反应温度是在 37℃。酶切反应时间有 30 分钟、60 分钟，以至 2 小时以上甚至过夜。本次实验要进行的是对重组质粒 pET-22b 进行 EcoRI 酶切，重组质粒上有两个 EcoRI 识别位点。
验步骤及操作方法
重点:
掌握细菌基因组 DNA 的提取的实验步骤及操作方法
难点: 掌握细菌基因组 DNA 的提取的实验步骤及操作方法
实验三. 大肠杆菌感受态细胞的制备及转化实验（4 学时）基本要求:
了解大肠杆菌感受态细胞的制备原理及转化反应的目的，应用范围及操作过程，掌握重组 DNA 质粒转化大肠杆菌的操作技术。重点:
实验步骤
PCR 反应的原理
1 在 02ml 离心管管内配制 50μL 反应体系：
（1）ddH2O
38μL
（2）10×buffer
5μL
（3）dNTP
1μL
（4）Taq 酶
1μL
（5）引物 P1
1μL
（6）引物 2
1μL
（7）模板 DNA
3μL
2 按下列程序进行扩增：
（1） 94℃预变性 5 分钟；（2） 94℃变性 50 秒；
基本要求：了解分子生物学的实验操作原理，掌握有关实验仪器的使用方法。
实验内容：
实验一.细菌质粒 DNA 的提取及酶切实验
（8 学时）
基本要求:
通过学习碱变性抽提法对大肠杆菌中的质粒的抽提，掌握质粒 DNA 的小量
制备方法，了解碱裂解法制备质粒 DNA 的原理。进一步了解限制性
内切酶的特性及酶切反应过程，掌握 DNA 的酶切技术。
质粒图谱 1
质粒图谱 2
实验步骤
1 收集菌泥。
2 悬浮细菌。
3 加入 200μl 裂解液 (Solution 2)，充分裂解菌体。
4 加入 400μl 絮凝剂(Solution 3)。
5 15000rpm 离心 10 分钟，无需低温离心。
6 将步骤 5 中的上清转移到吸附柱 (3S) 中，
实验步骤：
1 将配好的电泳缓冲液，倒入电泳槽。 2 将琼脂糖凝胶放入微波炉熔化。 3 熔化后的琼脂糖凝胶倒入插好梳子的模具中。 4 静置 1 小时左右，等琼脂糖凝胶凝固后垂直拔去梳子。 5 将琼脂糖凝胶放入电泳槽中。 6 点样,一般上样量为 10×Loading Buffer 05-1μl、DNA 样品（质粒）3μl 混匀，点入凝胶孔内。 7 质粒电泳参数：电泳仪的电压 100V，电流 500mA，功率 250W。电泳时间 40 分钟。 8 将琼脂糖凝胶取出，放入紫外与可见分析装置。 9 凝胶成像系统显像，拍照。
重点: 掌握掌握琼脂糖凝胶电泳的实验方法。
难点: 聚合酶链反应（PCR）的实验技术，琼脂糖凝胶电泳的实验方法。
实验五. 聚丙烯酰氨凝胶电泳（SDS-PAGE）分离蛋白质（4 学时）基本要求:
了解聚丙烯酰氨凝胶电泳（SDS-PAGE）分离蛋白质的基本原理，学会用该法测定蛋白质分子量的操作技术。
重点: 掌握聚丙烯酰氨凝胶的配制方法，了解聚丙烯酰氨凝胶电泳（SDS-PAGE）分
掌握大肠杆菌感受态细胞的制备及转化的操作技术
难点: 大肠杆菌感受态细胞的制备及转化
实验四. 聚合酶链式反应（PCR）扩增目的基因及琼脂糖凝胶电泳（8 学时）基本要求:
熟悉聚合酶链反应（PCR）的基本原理、实验基本条件；了解 PCR 反应条件的优化和注意事项和应用范围掌握聚合酶链反应（PCR）的实验技术：了解核酸琼脂糖凝胶电泳的原理及操作步骤，掌握琼脂糖凝胶电泳的实验方法。
12000rpm 离心 1 分钟。
7 将吸附柱放入同一支收集管中，吸取洗涤液到吸附
柱，离心 1 分钟。
8 重复步骤 7 一次。 9 将吸附柱放入同一支收集管中，高速离心 2 分钟。
质粒电泳图
10 在吸附柱(3S)膜中央加 50μl 无菌双蒸水，室温高速离心 1 分钟。
实验三琼脂糖凝胶电泳
实验目的
生物工程与技术基础实验与工程实训中心
分子生物学实验室
实验一氯化钙法大肠杆菌感受态细胞的制备及转化实验二细菌质粒 DNA 的提取实验三琼脂糖凝胶电泳实验四细菌基因组 DNA 的提取与琼脂糖凝胶电泳实验五质粒 DNA 限制性内切酶实验实验六聚合酶链式反应（PCR）扩增目的基因实验七胶回收法纯化 DNA 与琼脂糖凝胶电泳
仪器名称型号
标准型双人净SW-CJ-IC
标准型双人净SW-CJ-IC
冰箱
海尔BCD-210
超声波细胞粉JY92-II2D
电热鼓风干燥101C-3
电子天平 BS224S
（3） 55℃退火 50 秒；（4） 72℃延伸 10 秒；
（5）重复步骤②-④30 次；
（6） 72℃彻底延伸 10 分钟。
琼脂糖凝胶电泳图
3 琼脂糖凝胶电泳分析 PCR 结果。
实验七胶回收法纯化 DNA
实验目的
本实验利用胶回收纯化试剂盒从琼脂糖凝胶中将目的 DNA 片段进行抽提纯化，从中掌握切胶技术以及回收目的 DNA 片段并纯化的方法。
大肠杆菌的细胞的结构
实验步骤
细菌基因组 DNA 的结构
1 细菌的消化 2 加入 400 ul DL 液和 25 ul 蛋白酶 K，迅速温和地来回颠倒离心管彻底混匀。
温浴 30 分钟。离心 1 分钟后，取上清至另外一个离心管中。 3 加入 200 ul 异丙醇,离心弃去收集管中的液体。 4 加入 500 ul 洗涤液，离心 30 秒。 5 将吸附柱移入另外一个干净的收集管中。离心 15 秒。 6 将吸附柱放入同一个收集管中。离心 1 分钟。 7 在吸附膜中央加入 100 ul T1 液，离心 1 分钟。将 DNA -20 ℃ 保存。 8 取纯化后基因组片段洗脱液 2μl 加上样缓冲液 1μl 混匀，标准分子量 DNA2-3 μl，电泳检测结果。