实验二基因的PCR扩增技术

实验二基因的PCR扩增技术 一、实验目的 1、学习PCR反应的基本原理与实验技术 2、从BCG基因组DNA中PCR扩增Rv1791(PE19)基因 二、基本原理 1、PCR类似于DNA的天然复制过程。 2、将待扩增的DNA片段与其两侧互补的两段寡核苷酸引物，经变性、 n退火和延伸若干个循环后，DNA扩增倍数可达2倍。 3、DNA的变性和复性 1)加热或强酸、碱性作用可以使 DNA双螺旋的氢键断裂,双链解离,形成单链DNA,这称为 DNA的变性。 2)解除变性的条件后, 变性的单链可以重新结合起来,形成双链,其原有的特性和活性可以恢复,这称DNA复性, 也叫退火。 三、实验材料 , BCG基因组DNA , 10，PCR反应缓冲液、4，dNTPs、Taq DNA聚合酶、模板DNA、引 物 (P1、P2)、超纯水SW , PCR反应管、移液器、 PCR仪、离心机 四、实验步骤 , 引物 , 准备PCR反应溶液 , PCR扩增反应 the three. Third, seriously implement the Central, provincial, municipal and County party Committee on the establishment of a sound work style construction views of the long-acting mechanism and on the implementation of the three Suns calls for further strengthening of education administration and supervision of party members and cadres, from the ... Accept supervision and earnestly grasp, balance, two promotion, important job himself, the Secretary of development, major issues personally. 5. strongly supports accountability discipline discipline. Administrative provisions on strict enforcement of party members and cadres to ask all the discipline inspection and supervision cadres in accordance with the iron itself must be a hard requirement, adhere to the responsibilities and play, earnestly fulfill their duties of supervision and accountability for Zhi JI. Zhen into full play and the oversight role of the Commission, establish and improve the village rules and community conventions, promoting joint public and serving the sunshine publicly available implementation of the platform for action. Discipline in handling cases, adhere to find along with scrutiny, to appoint the interference and resistance, backed by courageous, find the problem corrected, powerful maintenance the seriousness of responsibility of uncorrupted. Actively promote the discipline Committee detailed switch and, casting, tree-generating activities. Promote , 结果 , Rv1791-F: ATGAATTCAGGAAGACAGCATGTCGTTC(EcoR I) , Rv1791-R: , ATCTCGAGTGTTCCCCCTCTCCTTCAG , (XhoI) (二)准备PCR反应溶液 10×PCR反应缓冲液 2.5μl 4×dNTPs 2.0μl 引物P1 1μl(10nM) 引物P2 1μl 模板DNA 2μl Taq DNA聚合酶 0.2μl 用无菌去离子水至 25μl ? 用手指轻弹PCR反应管管底部，使溶液混匀 ? 在台式离心机中离心2秒以集中溶液于管底 (三)PCR扩增反应 在PCR仪中设计如下反应程序:94:C、5min;94:C、30sec，61:C、40sec，72:C、30sec，30个循环;72:C、5min 将已混匀的PCR反应管置于PCR仪的反应孔中，盖好盖子 进行反应。反应完毕，将样品取出置于冰浴中待用 the three. Third, seriously implement the Central, provincial, municipal and County party Committee on the establishment of a sound work style construction views of the long-acting mechanism and on the implementation of the three Suns calls for further strengthening of education administration and supervision of party members and cadres, from the ... Accept supervision and earnestly grasp, balance, two promotion, important job himself, the Secretary of development, major issues personally. 5. strongly supports accountability discipline discipline. Administrative provisions on strict enforcement of party members and cadres to ask all the discipline inspection and supervision cadres in accordance with the iron itself must be a hard requirement, adhere to the responsibilities and play, earnestly fulfill their duties of supervision and accountability for Zhi JI. Zhen into full play and the oversight role of the Commission, establish and improve the village rules and community conventions, promoting joint public and serving the sunshine publicly available implementation of the platform for action. Discipline in handling cases, adhere to find along with scrutiny, to appoint the interference and resistance, backed by courageous, find the problem corrected, powerful maintenance the seriousness of responsibility of uncorrupted. Actively promote the discipline Committee detailed switch and, casting, tree-generating activities. Promote (四)结果检测 , 本PCR扩增的产物DNA片段长度为300bp，适合于在1.5%琼脂 糖凝胶中进行电泳检测。从每个反应管中取5ul PCR产物进行 电泳检测。 五、实验结果与分析 本组实验序号为41号。 Rv1791(PE19)基因的分子量在250bp-500bp之间，符合已知数据DNA分子量是300bp，PCR扩增成功。本组实验PCR扩增的DNA量较少，可能原因有实验一时提取的DNA含量较少，DNA未与PCR反应溶液混合均匀。 六、思考题 , 影响PCR结果的因素。 解: 1.外部因素对实验结果的影响: the three. Third, seriously implement the Central, provincial, municipal and County party Committee on the establishment of a sound work style construction views of the long-acting mechanism and on the implementation of the three Suns calls for further strengthening of education administration and supervision of party members and cadres, from the ... Accept supervision and earnestly grasp, balance, two promotion, important job himself, the Secretary of development, major issues personally. 5. strongly supports accountability discipline discipline. Administrative provisions on strict enforcement of party members and cadres to ask all the discipline inspection and supervision cadres in accordance with the iron itself must be a hard requirement, adhere to the responsibilities and play, earnestly fulfill their duties of supervision and accountability for Zhi JI. Zhen into full play and the oversight role of the Commission, establish and improve the village rules and community conventions, promoting joint public and serving the sunshine publicly available implementation of the platform for action. Discipline in handling cases, adhere to find along with scrutiny, to appoint the interference and resistance, backed by courageous, find the problem corrected, powerful maintenance the seriousness of responsibility of uncorrupted. Actively promote the discipline Committee detailed switch and, casting, tree-generating activities. Promote 不同PCR仪品牌与型号在温度控制性能上的不同直接影响了三种温度平台的温度的准确性和不均一性，因而影响了整个PCR反应的实验结果。另外，升温速度的不同，温度下降梯度的不均一性，温度过低，温度过冲，也是重要的因素。最差的情况，一个本该阳性的结果却可能得出阴性的结果。对于实时PCR，荧光信号输出和产量/溶解曲线，直接与温度控制有关系，而这些结果很可能是戏剧性的。 2.PCR仪器之间的差别对实验结果的影响: 没有任何两台PCR仪器的温度控制是一模一样的。相对于预设的温度模块温度总是偏高或偏低，就是同一模块不同的孔之间，温度有时也会不一样，相差可能达几摄氏度之多。一些PCR仪温度控制性能的不足造成的后果是:当PCR仪的模块温度在升温时，温度过冲或不足，不能准确的达到预设的平台温度。 3.温度控制技术的差别对实验结果的影响: 使PCR仪模块的温度达到并保持在某一温度有很多方式。每一种方式都有优点和缺点。另外，这些技术被运用控制的水平对于温度控制的水准是很重要的，且有很大不同。大多数PCR仪器的温度控制性能是很差的。在单一或多传感器控制机制上的不同显而易见的引起了温度的不均一性。 4.仪器老化对实验结果的影响: 虽然PCR仪没有任何的机械或可移动部件，但是那并不意味着它不会磨损。电子器件的性能会随着时间的延长而老化，并由于过度的使用而加速老化。Peltier器件的失效或磨损会引起模块的热斑或冷the three. Third, seriously implement the Central, provincial, municipal and County party Committee on the establishment of a sound work style construction views of the long-acting mechanism and on the implementation of the three Suns calls for further strengthening of education administration and supervision of party members and cadres, from the ... Accept supervision and earnestly grasp, balance, two promotion, important job himself, the Secretary of development, major issues personally. 5. strongly supports accountability discipline discipline. Administrative provisions on strict enforcement of party members and cadres to ask all the discipline inspection and supervision cadres in accordance with the iron itself must be a hard requirement, adhere to the responsibilities and play, earnestly fulfill their duties of supervision and accountability for Zhi JI. Zhen into full play and the oversight role of the Commission, establish and improve the village rules and community conventions, promoting joint public and serving the sunshine publicly available implementation of the platform for action. Discipline in handling cases, adhere to find along with scrutiny, to appoint the interference and resistance, backed by courageous, find the problem corrected, powerful maintenance the seriousness of responsibility of uncorrupted. Actively promote the discipline Committee detailed switch and, casting, tree-generating activities. Promote 斑，通常PCR仪器不能识别这种由于机械磨损，模块延展和收缩效应引起的温控性能的不同。因此，为掌握这些情况，实时监测PCR仪的温度控制性能是重要的。出于同样的原因,PCR仪温控性能的失常通常是实验失败或毁坏循环反应的重要原因。 5.实验室认证: 根据以上的论点，很多实验室已经不得不适应一些认证。和ISO、GMP、GLP一样，必须证明所有的实验室程序是按照标准程序所操作的。一些实验室只有当他们采用已经被他们的合作组织或专业委员会测试的或认可的程序时才可以称他们自己是合格的。 the three. Third, seriously implement the Central, provincial, municipal and County party Committee on the establishment of a sound work style construction views of the long-acting mechanism and on the implementation of the three Suns calls for further strengthening of education administration and supervision of party members and cadres, from the ... Accept supervision and earnestly grasp, balance, two promotion, important job himself, the Secretary of development, major issues personally. 5. strongly supports accountability discipline discipline. Administrative provisions on strict enforcement of party members and cadres to ask all the discipline inspection and supervision cadres in accordance with the iron itself must be a hard requirement, adhere to the responsibilities and play, earnestly fulfill their duties of supervision and accountability for Zhi JI. Zhen into full play and the oversight role of the Commission, establish and improve the village rules and community conventions, promoting joint public and serving the sunshine publicly available implementation of the platform for action. Discipline in handling cases, adhere to find along with scrutiny, to appoint the interference and resistance, backed by courageous, find the problem corrected, powerful maintenance the seriousness of responsibility of uncorrupted. Actively promote the discipline Committee detailed switch and, casting, tree-generating activities. Promote