DNA实验技术

一、碱变性法提取质粒DNA 质粒(Plasmid)是细菌染色体外能自身独立复制的双股环状DNA。带有遗传信息，可赋予细菌某些新的表型。将质粒指纹图谱分析方法、质粒 DNA探针技术及检测质粒的PCR技术用于临床感染性疾病的诊断和流行病学调查已成为现实。质粒作为载体在基因工程中起着重要的作用。分离和纯化质粒DNA的方法很多但这些方法基本包括三个步骤：即细菌的培养和质粒DNA的扩增；细菌菌体的裂解;质粒 ...

一、实验材料1、宿主细胞CHO（贴壁细胞）2、脂质体LIPOFECTAMINE 2000（invitrogen公司）3、6孔细胞培养版4、无血清培养基OPTI-MEM（GIBICO）5、转染级质粒二、实验步骤invitrogen的LIPOFECTAMINE 2000说明书上列举了24孔、12孔、6孔......板的实验体系，因为需要转染的细胞量大，所以一直采用的是6孔版做的转染。以下是以6孔板为例 ...

1. Transfer 5-10 μg of poly(A)+ RNA to a sterile microfuge tube. Adjust the volume of the solution to 40 μl with RNase-freeH2O. Heat the closed tube to 70°C for 5 minutes and then quickly transfer the ...

大肠杆菌质粒DNA的提取（碱裂解法）此方法适用于小量质粒DNA的提取提取的质粒DNA可直接用于酶切PCR扩增。1 ). 取1.5ml细菌培养物于EP管中，4000rpm离心1分钟，弃上清液，使细菌沉淀尽量干燥；2 ). 将细菌沉淀重悬于用冰预冷的100 µl溶液I (50 mmol/L葡萄糖，10 mmol/L EDTA pH 8.0，25 mmol/L Tris-HCl pH 8.0 ...

Phenol extraction is a common technique used to purify a DNA sample (1). Typically an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vi ...

After lysing the samples. DNA or RNA needs to be purified. If it is RNA sample the lysis buffer must contain something (2-merceptoethanol) that will destroy all RNAse enzyme activity.Then you can puri ...

碱裂解法从大肠杆菌制备质粒，是从事分子生物学研究的实验室每天都要用的常规技术可是我收研究生十几年了，几乎毫无例外的是我那些给人感觉什么都知道的优秀学生却对碱法质粒抽提的原理知之甚少追其原因，我想大概是因为分子克隆里面只讲实验操作步骤，而没有对原理进行详细的论述这是导致我的学生误入歧途的主要原因后来我发现其实是整个中国的相关领域的研究生水平都差不多，甚至有很多老师也是这个状态这就不得不让人感到悲哀了 ...

T4噬菌体DNA连接酶不同于大肠杆菌DNA连接酶，它可以催化平端DNA片段的连接（Sgaramella和Khorana1972;Sgaramella和Ehrlich1978），由于DNA很容易成为平端，所以这是一个极为有用的酶学物性。有了这样的物性，才能使任何DNA分子彼此相连。优点：1、没有连不上的，只有切不开的。平末端连接不牵扯到粘性末端的碱基突出问题，所以，理论上任何两条DNA序列都能连接在 ...

质粒是携带外源基因进入细菌中扩增或表达的主要载体，它在基因操作中具有重要作用。质粒的分离与提取是最常用、最基本的实验技术。质粒的提取方法很多，大多包括3个主要步骤：细菌的培养、细菌的收集和裂解、质粒DNA的分离和纯化。本实验以碱裂解法为例，介绍质粒的抽提过程。实验目的：掌握碱裂解法抽提质粒的原理、步骤及各试剂的作用。实验材料：含有质粒pUC18载体的大肠杆菌菌液，克隆有水稻外源片段的BAC的大肠杆 ...

质粒的转化是指将质粒或以它为载体构建的重组子导入细菌的过程。将连接产物转化到感受态细胞中，实现重组克隆的增殖，便于后续分子操作。可以采用多种方法筛选和鉴定目的克隆。实验目的：掌握热激法或电转化法转化大肠杆菌感受态细胞及转化子的鉴定方法。实验材料：外源片段与载体的连接产物；大肠杆菌感受态细胞。实验原理：（1）热激法：大肠杆菌在0℃ CaCl2低渗溶液中，菌细胞膨胀成球形，转化混合物中的DNA形成抗D ...

There are multiple variations to this protocol but we find that this one works well in all cases we tested.Reagents:5X EMSA Buffer:50mM HEPES (pH 7.9)375 mM KCl12.5 mM MgCl20.5 mM EDTA5 mM DTT15% Fico ...

To prepare polyacrylamide gels for DNA sequencing the appropriate amount of urea is dissolved by heating in water and electrophoresis buffer the respective amount of deionized acrylamide-bisacrylamide ...

Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions are modified from those presented earlier by altering the absolute amounts and the relative deoxy/dideoxynucleotide ratios i ...

Each base-specific fluorescent-labeled cycle sequencing reaction routinely included approximately 100 or 200 ng Biomek isolated single-stranded DNA for A and C or G and T reactions respectively. Doubl ...

One of the major problems in DNA cycle sequencing is that when fluorescent primers (1) are used the reaction conditions are such that the nested fragment set distribution is highly dependent upon the ...

Single-stranded dye-terminator reactions required approximately 2 ug of phenol extracted M13-based template DNA. The DNA is denatured and the primer annealed by incubating DNA primer and buffer at 65d ...

Polyacrylamide gels for fluorescent DNA sequencing are prepared as described above except that the gel mix is filtered prior to polymerization. Optically-ground low fluorescence glass plates are caref ...

Sequencing double stranded DNA templates has become a common and efficient procedure (10) for rapidly obtaining sequence data while avoiding preparation of single stranded DNA. Double stranded templat ...

The following is a rapid and efficient method for sequencing cloned cDNAs based on PCR amplification (14) random shotgun cloning (1315) and automated fluorescent sequencing (16). This method was devel ...

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螺旋线形开环，线形的质粒在中间，而开环的质粒在最后。判断质粒质粒好坏的一个指标就是超螺旋质粒在所以质粒中的含量。因为用质粒转染真核细胞时，超螺旋的质粒效率最高，所以要求质粒中超螺旋质粒的含量要在90％以上，这也是对作为核酸疫苗 ...