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生化与分子生物学分子部分.ppt

1、生化与分子生物学实验,概述,实验设计:Get to know the protein you want to cloneAcquire the coding sequence of the proteinDecide the vector you want to useDesign primers for coding sequence PCRPrepare PCR templatesPCR and prepare insertion fragment (digestion )Prepare vector fragments (digestion, purification)Ligation,

2、 transformation, screening, verificationTransfer expression plasmid to expression host cellsExpression, purification, characterization of cloned protein,生化与分子生物学实验I,实验用试剂的配制.器皿的准备及灭菌,实验内容(以下内容均按每小组4人计算),1)每小组共配制100ml为固体培养基, 装在250ml锥型瓶中; 高压灭菌(121/20min), 然后4放置备用。 LB固、液体培养基配方(按1L计算) Tryptone 10g (1%)

3、Yeast Extract 5g (0.5%) NaCl 10g (1%) 加蒸馏水溶解,用NaOH调pH值至7.0,定容到1000ml。 固体培养基加琼脂粉浓度为1.5%(W/V),2). 20ml 0.1M CaCl2 ( MW:111)3). 培养皿(4块) 包裹,4). 大(蓝色)和小(黄色)枪头各2盒5). 1.5ml Eppendorf管一烧杯 灭菌(121/20min),实验 一. pTKD GI质粒 DNA的提取、与酶切,l.1 质粒DNA的提取,原理:质粒(plasmid)是一种染色体外的稳定遗传因子,大小在1一2OOkb之间,具有双链闭合环状结构的DNA分子,主要发现于细菌

4、、放线菌和真菌细胞中。质粒具有自主复制和转录能力,能使子代细胞保持它们恒定的拷贝数,可表达它携带的遗传信息。它可独立游离在细胞质内,也可以整合到细菌染色体申,它离开宿主的细胞就不能存活,而它控制的许多生物学功能也可对宿主细胞的相应缺陷进行补偿。,本实验分离纯化的质粒为插入一个外源GI基因到 pTKD 质粒中的 pTKD GI质粒, 长度大约为 4.3 + 1.2 (kbp). 所有分离质粒DNA的方法都包括三个基本步骤: 1.培养细菌使质粒扩增; 2.收集和裂解细菌; 3.分离和纯化质粒DNA。,离心机的正确使用: 平衡: 在转子的对称位置的管子 等重量,小的台式离心机则仅要求对称位置2管要等

5、体积,大肠杆菌质粒DNA的分离及纯化(碱变性法),试剂:1. Solution I (GET): 5O mmol/L 葡萄糖, lO mmoI/L EDTA-Na2( pH8.0) 25 mmol/L Tris-HCl ( pH8.0)2. Solution II: 0.2mol/L NaOH 1% SDS3. Solution III 60ml 5mol/L KAc 11.5ml HAc 28.5ml dH2O 最终: Ac-=5mmol/L K+=3mmol/L pH=4.8,4. TE buffer(pH8.0) 10mmol/L Tris-HCl (pH8.0) 1mmol/L EDT

6、A-Na2 (pH8.0)RNase A 10mg/ml in dH2O苯酚/氯仿/异戊醇 V/V 25:24:1, 用前需用TE buffer 饱和, 取用下层有机相. (4度避光保存)氯仿/异戊醇 V/V 24:1 (4度保存)3mol/L NaAc (pH5.2)无水乙醇, 70%乙醇 (-20度保存),大肠杆菌质粒DNA的分离及纯化(碱变性法),Protocol于3 ml LB液体培养基(内含相应的抗生素)中接 种单菌落,37振荡培养过夜;2. 吸取细菌培养液置于Eppendorf管中, 12500rpm离心30 sec,弃尽上清; 3. 加入4预冷的 Solution I 100ul

7、,充分悬浮菌体,同时加入RNase (10mg/ml) 2 ul,充分混匀, 室温5-10min;4. 加入200ul新配的Solution II,轻轻颠倒数次充分混匀,冰浴5-10min至变清亮;,5. 加入4预冷的 Solution III 150ul,反复颠倒充分混匀,冰浴10min;6. 12500rpm离心5-7min,上清移至新Epp管中;7. 加入200ul苯酚/氯仿/异戊醇 (25:24:1),剧烈 振荡,12500rpm离心5min, 回收上层水相至新Epp管中;8. 加入200ul氯仿/异戊醇 (24:1),剧烈振荡, 12500rpm离心5min, 回收上层水相至新管中;

8、9. 加入1/10体积3M NaAc (pH5.2), 2-2.5倍体积 冰无水乙醇,混匀,-2010min;,10. 12500rpm离心15min,弃上清;用400 ul 70%冰乙醇洗涤沉淀,12500rpm离心3min,弃尽上清; 11.室温或37温箱干燥后,用30 ul ddH2O/TE (灭菌)溶解沉淀,4保存备用。,操作步骤:用碱裂解法1.培养细菌 将带有质粒的大肠杆菌接种在LB液体培养基,37oC培养12-18h。质粒提取方法请按下方法进行(一种不用酚与氯仿的质粒提取法,减少环境污染,也更安全)。,1、取1.5 ml O.N.菌液于Ep管中,转速125OO rpm离心30秒,去

9、净上清液。请注意离心机配平2、完全悬浮菌于150 L 冰冷的Solution I缓冲液。(振荡器)3、加入200 L新配制的Solution II(0.2 mol/L NaOH ,1% SDS)。盖紧,快速颠倒混匀直至液体变清(较粘)。4、加入15OL冰冷的Solution III,盖紧,颠倒数次至液体完全混合,冰浴5分钟。5、125OO rpm离心裂解液10分钟,转移尽量多的上清到新Ep管。6、上清中加入400 L异丙醇,盖紧,快速混匀,室温放置5分钟后, 125OO rpm离心5分钟,弃上清。注意沉淀不要倒掉7、加入200 L ddH2O(灭菌)溶解沉淀,再加入100 L 7.5M NH4

10、Ac,混匀后冰上放置5分钟8、12500 rpm 离心5分钟,转移尽量多的上清到新Ep管。加入600 L 无水乙醇,混匀后 20 oC 放置 20分钟。,6、上清中加入400 L异丙醇,盖紧,快速混匀,室温放置5分钟后, 125OO rpm离心5分钟,弃上清。注意沉淀不要倒掉7、加入200 L ddH2O(灭菌)溶解沉淀,再加入100 L 7.5M NH4Ac,混匀后冰上放置5分钟8、12500 rpm 离心5分钟,转移尽量多的上清到新Ep管。加入600 L 无水乙醇,混匀后20 oC 放置 20分钟。 9、125OO rpm离心5分钟,弃上清。注意沉淀不要倒掉,加入 1ml 70乙醇颠倒润洗

11、,冰上放置5分钟。 10、 125OO rpm离心5分钟,弃净上清,注意沉淀不要倒掉。开盖室温放置直至乙醇完全挥发。11、溶解DNA沉淀至20 L TE(灭菌)缓冲液中。,1.2 质粒的酶切,本次实验所用的质粒酶切图选用EcoRI 和BamHI 进行双酶切,Linearize plasmids with restriction enzyme,Digest foreign DNA with same enzyme if 6 base recognition site is random in genome: cuts every 4096 bp (46) human genome = 3 x 1

12、09 bp7 x 105 fragments,single fragments are ligated to individual plasmid DNAs transformation introduces individual plasmids (clones) into bacteria,Entire population of transformants represents genomic library,Restriction Endonucleases: have a specific recognition sequence (e.g 4-6 bp) many enzymes

13、have palindromic recognition sites DNA cut with these enzymes is broken within the strand some of the enzymes leave a single strand overhang (sticky end),Setting Up a Restriction Endonuclease Reaction,1. A Typical Restriction Digest,1ul of enzyme 1 ug of purified DNA 50 ul of the appropriate 1X comp

14、actable Buffer incubate for 1 hour at the 37 C,Setting Up a Restriction Endonuclease Reaction,2. Choosing the Right Enzyme,the DNA to be digested must contain a recognition sequence for the restriction enzymes chosen. No extra recognition sequence within your target fragment,Setting Up a Restriction

15、 Endonuclease Reaction,3. Enzyme Should be kept on ice when they are not in the freezer. Should be out of freezer as little time as possible.3. Should always be the last component added to a reaction mix,Setting Up a Restriction Endonuclease Reaction,4. DNAshould be free of contaminants such as phen

16、ol, chloroform, alcohol, EDTA, detergents, or excessive salts. DNA methylation is also an important element of a restriction digest.,Setting Up a Restriction Endonuclease Reaction,5. Reaction Buffer Each enzyme needs a specific buffer to ensure optimal (100%) activity. Buffer are usually supplied at

17、 10X concentrationThe buffer should be used at 1X concentration in the reaction. Some restriction enzymes require bovine serum albumin (BSA),Setting Up a Restriction Endonuclease Reaction,6. Reaction Volume1 unit of restriction enzyme will completely digest 1 g of substrate DNA in a 50 l reaction in

18、 60 minutes. This enzyme : DNA : reaction volume ratio can be used as a general guide. glycerol concentration should be less than 5% in a reaction, the restriction enzyme, which is supplied in 50% glycerol, should not exceed 10% of the total reaction volume.,Setting Up a Restriction Endonuclease Rea

19、ction,7. Mixingreaction must be thoroughly mixed to achieve complete digestion. We recommend gently pipetting the reaction mixture up and down or “flicking” the reaction tube. Follow with a quick (“touch”) spin-down in a microcentrifuge. Do not vortex the reaction.,Setting Up a Restriction Endonucle

20、ase Reaction,8. Star activity: relaxed specificity, altered specificity,Setting Up a Restriction Endonuclease Reaction,Conditions that Contribute to Star ActivityHigh glycerol concentration 5% v/v High units to g of DNA ratio Varies with each enzyme, usually 100 units/g Low ionic strength pH 8.0 Pre

21、sence of organic solvents DMSO, ethanol (9), ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane (12) Substitution of Mg+ with other divalent cations Mn+, Cu+, Co+, Zn+,Setting Up a Restriction Endonuclease Reaction,Use as few units as possible to get a complete digestion. This avoids

22、overdigestion and reduces the final glycerol concentration in the reaction. Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation. Raise the ionic strength of the reaction buffer to 100-150 mM (provided the enzyme is not inhibited by hi

23、gh salt). Lower the pH of the reaction buffer to pH 7.0. Use Mg+ as the divalent cation.,Inhibiting Star activity,NEBuffer Activity Chart for Restriction Enzymes,Restriction endonucleases supplyer,New England Biolabs Stratagene Fermentas Takara Amershambiosciences 华美,20 l plasmid 2.5 l 10X multicore

24、 buffer 1l BamH1(2units) 1l EcoR1 (2units) 0.5 l ddH2Ototal volume 25l total Gentle 混匀(不能振荡),稍离心,37 C 酶解3小时,恒温箱。 20 C 保存,待用。,Reaction Mixture (切记:冰上操作!),1.3 DNA的琼脂糖凝胶电泳鉴定,原 理在本实验中,自制质粒DNA在电泳凝胶中呈现3条区带。 电泳后的位置次序是:超螺旋、线性、开环。电泳结果不能作为判断分子量大小的依据。,操作步骤:1、琼脂糖凝胶的制备 1%琼脂糖 in 1X TBE buffer ,煮化清亮后冷却到70度时可制胶,倒胶前

25、加入EB。(加 10mg/ml EB ,3ul/ 50ml胶)2、制板:凝胶的厚度在大约 0.60.7 厘米,不少于0.5 厘米4、加样:每孔加样约15ul (25ul 样品5ul 6X样品缓冲液)。 样品缓冲液含有:a. 缓冲系统, b. 蔗糖, c.染料指示剂。5、电泳:50V,0.51小时6、观察、分析TBE缓冲液配方: 10 X 贮液: 54 g Tris 碱 27.5 g 硼酸 20 ml 0.5 M EDTA, pH8.0 加水至500 ml.,标准分子量图谱,pETCAP 质粒,酶切,与PCR胶图 1 2 3 4 5,质粒DNA,超螺旋,RI、BamHI酶切载体片段,RI、Bam

26、HI酶切小片段,PCR扩增片段,Lane1. PlasmidLane2.RI, BamHI digestion of plasmidLane3. &4: PCRLane5. Marker,1.4 DNA样品的回收及连接,实验目的:利用凝胶回收试剂盒从凝胶中回收 目的DNA片段,并与特定的载体片 段进行连接反应,Why purify fragment?,Mixed digestion products can be used for ligation-making genomic libraryUse purified DNA fragments for purposed cloning-Mus

27、t know what you want before hand 1. simplify transforments screening procedures. 2. Concentrate target DNA fragment. 3. Improve ligation efficiency,Recover DNA from Gel (v-gene kit),用一灭菌刀片割下含目的DNA片段的琼脂块,尽量除去多余的不含DNA的胶切碎后放入Eppendorf管(60ul)加入300ul溶胶液(DE-A ),75水浴10分钟,使凝胶完全溶化,每2分钟颠倒混匀一次;加入150ul DE-B 溶液,

28、混合均匀后移入吸附柱,4000rpm 离心分钟,倒掉收集管中的液体,再将吸附柱放入同一收集管中;在吸附柱中加入500ul 洗涤液 (W1), 4000rpm离心30 秒,倒掉收集管中的液体,再将吸附柱放入同一收集管中;在吸附柱中加入700ul W2液,4000rpm离心30 秒, 倒掉收集管中的液体,再将吸附柱放入同一收集管中;重复步骤5一次再将吸附柱放入同一收集管中,12500rpm离心1分钟;将吸附柱放入一个灭菌的Epp管中(剪掉盖子),在吸附膜中央加入10 ul洗脱液 (灭菌TE/ddH2O),静置1分钟后,12500rpm离心1分钟,将含有DNA的Epp管于4放置备用。,Recover

29、 DNA from Gel (Sangon UNIQ-10 kit)用一灭菌刀片割下含目的DNA片段的琼脂块,尽量除去多余的不含DNA的胶切碎后放入Eppendorf管(60ul)加入 400ul Binding Buffer II,60水浴10分钟,使凝胶完全溶化,每2分钟颠倒混匀一次;将融胶液移入吸附柱,室温放置2分钟,8000 rpm 离心分钟,倒掉收集管中的液体,再将吸附柱放入同一收集管中;在吸附柱中加入500ul Wash Solution, 8000 rpm 离心1分钟, 倒掉收集管中的液体,再将吸附柱放入同一收集管中,重复步骤4一次再将吸附柱放入同一收集管中,12 500rpm离

30、心15秒;将吸附柱放入一个灭菌的Epp管中(剪掉盖子),在吸附膜中央加入10ul 60 预热的Elution Buffer(灭菌TE/ddH2O),室温 静置2分钟后,12 500rpm离心 1分钟,将含有DNA的Epp管于4放置备用。,1.5 Ligation,The formation of covalent phosphodiester bonds between 3-hydroxyl end of one nucleotide with the 5-phosphate end of another by DNA ligase.,Chemistry of ligation,DNA lig

31、ase,DNA ligase is a particular type of ligase (EC 6.5.1.1) that can link together DNA strands that have double-strand breaks,Commonly use: T4 DNA ligaseATP is needed,Mammalian ligase,DNA ligase I: ligates Okazaki fragments during lagging strand DNA replication and some recombinant fragments. DNA lig

32、ase II: alternatively spliced form of DNA ligase III found in non-dividing cells. DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing base excision mutations and recombinant fragments. DNA ligase IV: complexes with DNA protein XRCC4 and has similar function to DNA ligase III. I

33、t is more important in development.,Non-Homologous End Joining When both strands of the DNA break, and the cell has to glue it back together. It is an emergency repair system, error prone, still better than leaving the DNA in fragments. The Ku protein, and a DNA-dependent protein kinase (not shown)

34、are thought to bring the two ends together and hold them in place. DNA ligase, assisted by the Xrcc4 protein, seal the strands back together. The DNA ligase is shown as a schematic, since the atomic structure of the human form has not yet been solved.,Ligase active center,Controlling the optimal tem

35、perature,T4 DNA Ligase: most active at 25 The optimal enzyme temperature needs to be balanced with the Tm. of the DNA fragments being ligated. The shorter the DNA fragments, the lower the Tm. Thus for extremely short fragments on the order of tens of base pairs, ligation experiments are performed at

36、 very low temperatures (4 ) for overnight.,Make a circularized Plasmid,Vector: Replication Origin Screening marker Proper insertion sites(poly-linker)Target DNA fragment: Carry all the genetic information the you wish to be cloned. Have suitable sticky ends for ligation with vector DNA fragments.,Ch

37、oosing the Right Vector,Expression vec. vs. non expression vec.表达载体:Prokaryotic vs. eukaryotic 读码框,翻译起始密码,信号肽, 终止密码,片段插入方向等。非表达载体: Plasmid vec., YAC, BAC. Etc (the capability to carry differePnt sizes of insertion fragments.) There is no question of insertion orientation. Proper sites for Re-Cut to

38、recover DNA fragments. 不同的载体在宿主细胞中有不同的丰度,Ligation and recut,Perfectly matched restriction site sticky ends Can be re-cut with same restriction enzymeMatched sticky ends produced by different restriction enzymes: A. can be re-cut by a different third enzyme B. can not be re-cut by any enzyme.,设置连接反应的

39、对照管,16 过夜连接后,各管分别用于转化感受态细胞实际工作中一般载体片段的摩尔数可为载体片段的数倍(25倍)。(回收DNA亮度经验目测)反应体系中DNA的总终浓度约为10ng/ul平端连接时载体DNA可去磷酸化,减少载体自连。用的酶要多(5倍),Set up ligation reaction,连接反应体系为10ul,依次加入以下试剂(切记:冰上操作!) Vector fragment 2 ul Insert fragment 6 ul 10Ligation Buff 1 ul T4 DNA Ligase 1 ul Total Volume 10ul轻轻混匀,离心片刻, 12-16连接过夜。

40、,1.6 Making E.coli Transformation Competent Cell and Transformation With Ligated Plasmids,什么是转化(Transformation),转化(Transformation):是将外源DNA分子引入受体细胞,使之获得新的遗传性状的一种手段,它是微生物遗传、分子遗传、基因工程等研究领域的基本实验技术。自然发生的转化:质粒都可通过细菌接合作用转移到新的宿主内。转移必需的mob基因。在人工构建的质粒载体中:一般缺乏此种mob 基因,如需将质粒载体转移进受体细菌,需诱导受体细菌产生一种短暂的感受态以摄取外源DNA。,

41、受体菌的选择,转化过程所用的受体细胞一般是限制修饰系统缺陷的变异株,即不含限制性内切酶和甲基化酶的突变体(R,M),它可以容忍外源DNA分子进入体内并稳定地遗传给后代。一般是重组缺陷型,什么是感受态(Competent)细胞,受体细胞经过一些特殊方法(如电击法, CaCl2 , RbCl(KCl)等化学试剂法)的处理后, 细胞膜的通透性发生了暂时性的改变,成为能允许外源DNA分子进入的感受态细胞(Competent cells)。进入受体细胞的DNA分子通过复制,表达实现遗传信息的转移,使受体细胞出现新的遗传性状。在筛选培养基中培养,即可筛选出转化子(Transformants, 即带有异源D

42、NA分子的受体细胞)。,常用感受态细胞制备方法,CaCl2 :简便易行,且其转化效率完全可以满足一般实验的要求,制备出的感受态细胞暂时不用时,可加入占总体积15的无菌甘油于-70保存(半年),因此CaCl2法为使用更广泛。RbCl (KCl、MnCl2等 )法,制备的感受态细胞转化效率较高(10*810*9) , 方法较CaCl2法繁琐电穿孔法(Electroporation):又称电转化,转化效率高达10*10。多用于植物及酵母细胞的转化,转化率的计算,统计每个培养皿中的菌落数。在含抗生素的平板上长出的菌落即为转化子。转化子总数: 菌落数(筛选条件)稀释倍数转化反应原液总体积涂板菌液体积 转

43、化频率(转化子数每mg质粒DNA): 转化子总数质粒DNA加入量(mg) 感受态细胞总数: 对照组菌落数(非筛选条件)稀释倍数菌液总体积涂板菌液体积 感受态细胞转化效率; 转化子总数感受态细胞总数,CaCl2法制备感受态细胞,1于23 ml LB液体培养基中接种单菌落(DH5), 37振荡培养过夜;2取0.05ml培养物接种于50 ml LB 液体培养基中, 37摇瓶培养2-3 h至对数生长期(OD550=0.2-0.4);3 吸取1.5ml培养物至Epp管中,冰浴10min;4于4,4 000rpm离心10min,弃上清;5用0.5-1 ml冰预冷的0.1M CaCl2悬浮菌体,不能用震荡器

44、,用枪缓慢打匀。冰浴10-20 min;6于4,4 000rpm离心8min,弃上清;菌体用100ul 0.1M CaCl2悬浮,不能用震荡器,用枪缓慢打匀,立即用于转化或4短暂放置备用。,制备用于-70 保存的感受态细胞,基本步骤同前述,一般批量制备,可将50ml 菌液全用,使用试剂按比例放大。最后一步所用的CaCl2溶液中要预含有15的甘油。甘油的作用是减少冷冻时细胞内产生冰晶,减少细胞结构的破坏。用含甘油的CaCl2悬浮菌体后,按100ul份分装到EP管中,最好用液氮或乙醇干冰溶液急速冷冻后转入 -70 冰箱保存。-70 保存的感受态细胞在半年内可保证使用。每次现用现取,不能反复冻融。,

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