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载体（vector）是指运载外源DNA有效进入受体细胞内的工具。载体同外源DNA在体外重组成DNA重组分子，在进入受体后形成一个复制子，即形成在细胞内能独自进行自我复制的遗传因子。因此，作为载体应该满足以下几方面的要求：①有某种限制酶的一个切点，最好是有许多种限制酶的切点，而且每种酶的切点只有一个；②外源DNA插入后不影响载体在受体细胞中进行自我复制，载体应对受体细胞无害，以及载体能接纳尽可能大的 ...

脂质体(lipofectin regeant,LR)试剂是阳离子脂质体(DOTMA)和(DOPE)的混合物[1:1(w/w)]。它适用于把DNA转染入悬浮或贴壁培养细胞中，是目前条件下最方便的转染方法之一。转染率高，优于磷酸钙法，比它高5～100倍，能把DNA和RNA转染到各种细胞。

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一、质粒的提取及鉴定 （一）质粒的提取及鉴定 1、收获细菌 （1）将2ml含相应抗生素的LB液体培养基加入到通气良好的15ml的试管中，接入一单菌落，于37℃剧烈振荡培养过夜。 （2）将1.5ml培养物倒入1.5ml离心管中，用台式离心机于4℃以12000g离心5min，将剩余的培养物贮存于4℃。 （3）吸尽培养液，使细菌沉淀尽可能干燥。 2、碱裂解法小量抽提质粒 （1）将细菌沉淀重悬于100&m ...

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所谓亚克隆就是对已经获得的目的DNA片段进行重新克隆，其目的在于对目的DNA进行进一步分析，或者进行重组改造等。 亚克隆的基本过程包括：(1)目的DNA片段和载体的制备；(2)目的DNA片段和载体的连接；(3)连接产物的转化；(4)重组子筛选。 一、试剂准备 1．LB液体培养基：胰化蛋白胨（细菌培养用）10g，酵母提取物（细菌培养用）5g，NaCl10g，加ddH2O至1000ml，完全溶解，分装 ...

Materials: 3 M sodium acetate pH 5.2 or 5 M ammonium acetate DNA 100% ethanol Measure the volume of the DNA sample. Adjust the salt concentration by adding 1/10 volume of sodium a ...

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The preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonucleases. You will need: 10 x OPA+ Buffer (100mM Tris.acetate pH 7.5 100mM ...

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The gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA -binding proteins. This method has been used widely in the study of sequence-specific DNA -bi ...

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme in its respective buffer as recommended by the supplier and at ...

It is best to do a test ligation without insert if the background is low it would not be necessary to screen colonies for insert. 1.colony PCR 1) Pick a colony from plate and suspend the colony in 10 ...

简介 优点：操作简单方便 缺陷：不能像EMSA或footprinting一样区分不同类型复合物 原理 双链 DNA 能够通过硝化纤维膜 (NC)，但是 DNA-protein 复合物不能通过NC filter. 通过定量分析留在滤膜上的复合物，可以判断二者的结合常数。 基本方法 Lable DNA Mix DNA and protein to form complex Pass th ...

Sample Preparation Cheek cells are obtained by rinsing the mouth with 25mls of any commercial mouth wash solution available for about 30sec the first thing in the morning. It is important not to brus ...