实验讲义分子生物学

1、分子生物学实验讲义实验一、dna的分离纯化-碱裂解法提取质粒dna一、 实验目的：掌握碱裂解法提取质粒dna的原理与方法，了解各种试剂的作用。二、 实验原理：从细菌分离质粒dna的方法都包含3个基本步骤：培养细菌使质粒扩增；收集和裂解细胞；分离和纯化质粒dna。碱变性法抽提质粒是基于染色体dna与质粒dna的变性与复性的差异而达到分离目的。将细菌悬浮于葡萄糖等渗液中，经edta、naoh- sds溶液处理，可破坏菌体细胞壁和细胞膜，使细胞崩解。naoh破坏核酸碱基对使氢键断裂，细菌染色体dna和质粒dna变性，但质粒超螺旋共价闭合环状结构的两条互补链不会完全分离，所以当以ph4.8的kac高盐

2、缓冲液调节其ph至中性时，变性的质粒dna又恢复到原来的构型，重新形成天然的超螺旋分子，并以溶解状态存在于液相中。而线状染色体dna片段难以复性，与不稳定的大分子rna、蛋白质-sds-细胞膜复合物缠绕附着在细胞壁碎片上，冰浴后易沉淀，可离心除去。通过氯仿/异戊醇等有机溶剂除去蛋白质，再用乙醇等有机溶剂沉淀dna，即得纯化的质粒dna。三、 实验用品：如试剂: 溶液（葡萄糖维持渗透压、trishcl维持ph值、edta抑制核酸酶）50mmol/l葡萄糖 25mmol/ltriscl(ph8.0) 10mmol/ledta(ph8.0) 溶液（naoh裂解细胞与核酸变性、sds裂解细胞与蛋白质变

3、性）0.2mol/lnaoh（临用前用10mol/l贮存液现用现稀释） 1%sds 溶液（乙酸钾置换钠离子、冰乙酸调节ph值）5mol/乙酸钾60ml 冰乙酸11.5ml 水28.5ml 异丙醇、无水乙醇、蒸馏水四、 实验步骤：（一）细菌培养和收集将含有质粒的ecoli菌种接种在lb固体培养基（含50 g/ml amp）中，37oc培养1224小时。用无菌牙签挑取单菌落接种到5ml lb液体培养基（含50g/ml amp）中，37 oc振荡培养约12小时至对数生长后期。（二）质粒dna的少量快速提取（碱法）1取1.5ml培养液倒入1.5ml eppendorf管中，12000r/min 离心

4、1min。，弃上清，倒置片刻，使液体流尽。（可重复步骤1一次，以收集较多菌体）2菌体沉淀重悬于300l溶液中，剧烈振荡使菌体充分重悬，加入新配制的溶液300l，盖紧管口，立即将离心管缓慢颠倒数次直到溶液变清亮 (千万不要振荡)。3加入300l预冷的溶液，盖紧管口，缓缓颠倒离心管数次直到白色沉淀充分形成，冰浴5min，12000r/min离心5分钟。4上清液移入干净eppendorf管中，加等体积的氯仿/异戊醇（24:1），混匀，放置片刻。12000r/min离心5分钟。5将水相移入干净eppendorf管中，加入0.6倍体积异丙醇，震荡混匀后，12000r/min离心5分钟。6弃上清液，将管口

5、敞开倒置于卫生纸上，使所有液体流出，加入1ml 70%乙醇液沉淀一次，12000r/min离心5分钟。7吸除上清液，将管倒置于卫生纸上使液体流尽，真空干燥10分钟或室温干燥。8将沉淀溶于40-50l te缓冲液（ph8.0，含20g/ml rnasea）中，储于-20冰箱中。【注意】提取过程尽量保持低温。采用酚/氯仿去除蛋白效果较单独用酚或氯仿好。沉淀dna通常用冰乙醇，低温条件下放置时间稍长可使dna沉淀完全。五、实验结果：1、记录观察现象；2、分析原因如：质粒沉淀离心后,在管底能见到少量白色物质。真空干燥后，无色，贴于管壁。加ddh2o或te能迅速溶解。质粒中含太多杂质，则干燥后会呈白色或

6、褐色，不易溶解。实验二、核酸的检测（琼脂糖凝胶电泳）一、 实验目的：学习琼脂糖凝胶电泳分离dna的原理，掌握dna电泳分析技术。二、 实验原理：dna分子带负电荷，在电场中受到电荷效应、分子筛效应向正极移动过程中，因dna分子的大小及构象差别而呈现迁移位置上的差异，对于线形dna分子，其电场中的迁移率与其分子量的绝对值成反比。电泳时加溴化乙锭（eb），其与dna结合形成一种荧光络合物，在254-365nm紫外照射下可产生桔红色的荧光，可用于检测dna。三、 实验用品：四、 实验步骤：1、 琼脂糖凝胶板的制备： 1g琼脂糖50ml 电泳缓冲液，加热熔化均匀，冷却到6070，加10ul eb 混合

7、，倒入凝胶盒，厚35mm，插上点样梳，凝固后放入水平电泳槽，使电泳缓冲液淹没过胶12mm为止。2、 加样：取出点样梳，10ul样品2ul（吸嘴一小滴）上样缓冲液，混合后取10ul加入点样孔。同时设立marker（dna分子量标准物）3、 电泳：6090v，待溴酚蓝移至凝胶的2/3 距离时，停止电泳。4、 凝胶成像系统观察电泳结果。五、 实验结果：1、 画出电泳图。2、 简单说明分析。如：质粒dna在电场中带负电向正极移动，用酚、氯仿法从工程菌中提取的质粒一般有三种构像，即超螺旋，线形和开环。（开环质粒是指双链环状的质粒dna有部分解链，因此电泳速度最慢）三种构像的质粒在琼脂糖电泳的前后顺序是超

8、螺旋线形开环，线形的质粒在中间，而开环的质粒在最后。实验三、聚合酶链技术（pcr鉴定质粒）一、实验目的：1、熟悉pcr反应的基本原理；2、掌握pcr反应的操作方法。二、实验原理：体外快速的dna特异性扩增技术。模拟体内dna复制过程，通过变性、退火、延伸三步反应的循环完成，靶基因的双链dna变性为单链，特异的引物在退火过程中与单链dna模板聚合，在靶dna的指导下引物的3末端延伸靶dna的互补链，完成一个循环。理论上，每一循环使dna分子扩增一倍，n次循环后，dna分子扩增为2n；高温变性：94，目的基因变性为单链，以作为扩增的模板；低温复性：5560，一对特异性引物分别与模板3端互补结合；适

9、温延伸：72，延伸反应；本实验puc19质粒多克隆位点两侧含有恒定序列，用此序列为特异性引物进行pcr扩增，产物长度为158bp，以此鉴定puc19质粒。三、实验用品：四、实验步骤：1、50ul反应体系的配制，如表：ddh2o32.6ul10*buffer5ulmgcl2(25mm)待添加的隐藏文字内容35uldntp(10mm)1ulp1(10um)1ulp2(10um)1ultaq酶(5u/ul)0.4uldna模板4ul 充分混合，放入pcr仪扩增3、 反应条件：94.0 预变性 3min 94.0 30s 58.0 20s 30 cycles 72.0 5min 72.0 40s4、2

10、琼脂糖凝胶电泳检测（步骤同实验二）五、实验结果：1、画出电泳图。2、简单说明分析。winger tuivasa-sheck, who scored two tries in the kiwis 20-18 semi-final win over england, has been passed fit after a lower-leg injury, while slater has been named at full-back but is still recovering from a knee injury aggravated against usa.both sides boas

11、t 100% records heading into the encounter but australia have not conceded a try since josh charnleys effort in their first pool match against england on the opening day.aussie winger jarryd hayne is the competitions top try scorer with nine, closely followed by tuivasa-sheck with eight.but it is rec

12、ently named rugby league international federation player of the year sonny bill williams who has attracted the most interest in the tournament so far.the kiwi - with a tournament high 17 offloads - has the chance of becoming the first player to win the world cup in both rugby league and rugby union

13、after triumphing with the all blacks in 2011.id give every award back in a heartbeat just to get across the line this weekend, said williams.the (lack of) air up there watch mcayman islands-based webb, the head of fifas anti-racism taskforce, is in london for the football associations 150th annivers

14、ary celebrations and will attend citys premier league match at chelsea on sunday.i am going to be at the match tomorrow and i have asked to meet yaya toure, he told bbc sport.for me its about how he felt and i would like to speak to him first to find out what his experience was.uefa hasopened discip

15、linary proceedings against cskafor the racist behaviour of their fans duringcitys 2-1 win.michel platini, president of european footballs governing body, has also ordered an immediate investigation into the referees actions.cska said they were surprised and disappointed by toures complaint. in a sta

16、tement the russian side added: we found no racist insults from fans of cska. baumgartner the disappointing news: mission aborted.the supersonic descent could happen as early as sunda.the weather plays an important role in this mission. starting at the ground, conditions have to be very calm - winds

17、less than 2 mph, with no precipitation or humidity and limited cloud cover. the balloon, with capsule attached, will move through the lower level of the atmosphere (the troposphere) where our day-to-day weather lives. it will climb higher than the tip of mount everest (5.5 miles/8.85 kilometers), dr

18、ifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. as he crosses the boundary layer (called the tropopause),e can expect a lot of turbulence.the balloon will slowly drift to the edge of space at 120,000 feet ( then, i would as

19、sume, he will slowly step out onto something resembling an olympic diving platform.they blew it in 2008 when they got caught cold in the final and they will not make the same mistake against the kiwis in manchester.five years ago they cruised through to the final and so far history has repeated itse

20、lf here - the last try they conceded was scored by englands josh charnley in the opening game of the tournament.that could be classed as a weakness, a team under-cooked - but i have been impressed by the kangaroos focus in their games since then.they have been concentrating on the sort of stuff that wins you tough, even contests - strong defence, especially on their own goal-line, completing sets and a good kick-chase. theyve been great