DNA提取与纯化

Overview Procedure 1. Preheat the CTAB Isolation Buffer at 60℃. 2. Grind 2 g of fresh leaf tissue to a powder in Liquid Nitrogen in a chilled mortar and pestle. 3. Scrape the powder into a chilled 50 ...

1.溶液I―溶菌液: 溶菌酶:它是糖苷水解酶能水解菌体细胞壁的主要化学成分肽聚糖中的β-14糖苷键因而具有溶菌的作用。当溶液中pH小于8时溶菌酶作用受到抑制。 葡萄糖:增加溶液的粘度维持渗透压防止DNA 受机械剪切力作用而降解。 EDTA:(1)螯合Mg2+、Ca2+等金属离子抑制脱氧核糖核酸酶对DNA 的降解作用(DNase作用时需要一定的金属离子作辅基);(2)EDTA的存在有利于溶 ...

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Preparation of Silica 1. Suspend 5 g of silica (Sigma S-5631) in 50 ml of PBS. 2. Allow the silica to settle for 2h. 3. Discard the supernatant containing fine particulate matter. 4. Repeat steps 2 an ...

本方法通过SDS裂解细胞，蛋白酶降解蛋白，CTAB去除多糖成分 一:仪器:同方法一 二:试剂: TE、TAE缓冲液;10%SDS;5MNaCL; 20mg/ml蛋白酶K;CTAB/NaCL溶液(10% CTAB，0.7M NaCL 4.1gNaCL 溶于80ml水中，缓慢加入10gCTAB，加热溶解);25/24/1，酚/氯仿/异戊醇; 24/1，氯仿/异戊醇;异丙醇;70%及100 ...

1.Remove gel slice contain DNA fragment and place in 10 volumes of: 300 mM NaOAcpH 7.0 300 ml 1 M NaOACpH 7.0 1 mM EDTA 2 ml 500 mM EDTApH 8.0 698 ml ddH20 2.Incubate at 22℃ for 30 min.Transfer gel sl ...

(adapted from Bruce A.RoeDepartment of Chemistry and BiochemistryThe University of OklahomaNormanOklahoma 73019 broe@ou.edu) A.Large scale double-stranded DNA isolation The method used for the isolati ...

Recovery of DNA from LMP (Low Melting Point) Agarose Gel 1. Separate DNA fragments through an LMP agarose gel containing ethidium bromide (0.5 microgram/ml). 2. Detect DNA by irradiating the gel with ...

一、导论 质粒提取的原理:转自复旦大学一位老师的帖子后面是方法介绍 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 ...

Retroelements and their derivatives are a ubiquitous and abundant component of plant genomes. From the 1990s PCR based techniques have been developed to isolate the elements from genomic DNA of differ ...

Materials: • 0.8 % agarose gel in 1x TAE • Digested DNA • Glass Milk • NaI solution • New Wash Procedure: 1)Run digested DNA out on agarose gel slowly (70 V on BioRad gel) 2)U ...

1.Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube). 2.Resuspend pellet with 300μl STET buffer (900μl). After resuspending add 30μl RNase/lysozy ...

GEL PURIFICATION OF OLIGONUCLEOTIDES (Adapted from the method of Mark Fortini) 1. Quantitate crude oligo solution via UV spectrophotometry. Assume 1.0 A260 unit is equal to 33μl/ml. Dry down 100&mu ...

差减杂交法是目前广泛应用于差异性基因分离和鉴定的方法之一。简单地讲，差减杂交法是基于不同样品间特定核酸序列 ( 基因组 DNA 或 mRNA) 在数量 ( 等位基因数目或拷贝数或表达量 ) 上的差异，通过分子间的同源性杂交将存在数量差异性的序列分离出来。从研究对象和使用的基本材料来看，大致可以分为两类，即基因组差减杂交法和 mRNA 或 cDNA 差减杂交法。 1 ．原理 就是将不同来源的基因组D ...

一、实验目的 学习和掌握DNA的微量提取法。 二、实验原理 小麦黄化苗经液氮研磨，可使细胞壁破裂，加入去污剂（如SDS），可使核蛋白体解析，然后使蛋白和多糖杂质沉淀，DNA进入水相，再用酚、氯仿抽提纯化。本实验采用SDS法，其主要作用是破膜。SDS属阴离子去污剂，要溶解膜蛋白与脂肪，也可解聚核蛋白。SDS在溶液中带负电荷，能与带正电荷的蛋白质侧链结合成复合物，当加入钾盐时，能与SDS－蛋白质生 ...

一、目的: 了解质粒抽提常用的方法和原理；掌握试剂盒制备质粒DNA 的法。 二、原理： （一）质粒DNA 制备三个步骤： 1、细菌培养与质粒扩增 2、菌体收集和溶菌 3、质粒DNA分离 （二）方法介绍： 1、碱变性抽提法（SDS法）:主要是利用染色体DNA和质粒DNA碱性环境中变性和复性的差异来分离出质粒DNA。染色体DNA 分子量比较大，在碱性环境中（高pH）变性，双链解拆开，抽提时由于机械剪切 ...

Abstract: It is very difficult to isolate Rhizobium DNA due to the gum production by the organism. Hence we have designed a protocol for efficient isolation of DNA from the organism for further gene a ...

This is a four day procedure so it's best to start on Monday or Tuesday. CASE SELECTION: H&E stained thin sections are first reviewed by a pathologist and areas of interest are outlined. Tissue bl ...

Chapter 4 replication DNA replication DNA damage and repair Reverse transcription RNA replication DNA复制的基本特点 半保留复制(self-conservative replication) 复制子(replicon) 半保留复制 1958年Meselson 和Stahl首次用实验证实了DNA的半保 ...

Inoculate 1 ml of L-broth or other rich media with cells of the strain from which genomic DNA is to be isolated. Grow this culture at 37℃ overnight on a roller. Transfer this overnight culture to a 1. ...