DNA实验技术

真核细胞基因组提取 一. 实验目的及背景 高等动物，高等植物的基因组相当宠大， 如人类细胞基因组由30亿个碱基对组成，果蝇基因组有１．４×１０８个碱基对，水稻基因组有１．４×１０９个碱基对。真核细胞基因组中，约１万－１．５万个可表达的结构基因，其它大量存在的是调控序列和内含子序列。可以说某特定物种的基因组，包含了该物种生长，发育，繁殖等各项生理活动的几乎全部信息量，因而对真核细胞基因组的结构，组成 ...

背景知识:核酸的存在形式：脱氧核糖核酸(DNA)、核糖核酸(RNA) 与蛋白质结合在一起以核蛋白（DNP）的形式存在。在制备核酸时通常破坏细胞壁及细胞膜来使核蛋白释放出来。植物总DNA包括细胞核DNA和细胞核外DNA前者存在于细胞核内，后者存在于细胞质中有半自主性复制活性的细胞器内，例如线粒体DNA(mtDNA)和叶绿体DNA(ctDNA)。核酸的理化性质：DNA为白色类似石棉样的纤维状物， RN ...

复旦大学某教授写的有关质粒的好文，贴在这里与大家共享 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。可以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》试验方案 51protocol.com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 ...

(adapted from Bruce A. Roe Department of Chemistry and Biochemistry The University of Oklahoma Norman Oklahoma 73019 broe@ou.edu)Typically 2.5 - 3 volumes of an ethanol/acetate solution is added to th ...

This protocol yields plasmid DNA that is suitable for restriction digests and cloning purposes. This preparation method works well on all E. coli strains and also exponentially growing Rhizobium melil ...

(adapted from Bruce A. Roe Department of Chemistry and Biochemistry The University of Oklahoma Norman Oklahoma 73019 broe@ou.edu)Phenol extraction is a common technique used to purify a DNA sample (1) ...

【实验目的】 1．掌握核酸提取的基本原理和基本方法 2．掌握检测核酸浓度及纯度的原理及方法 【实验原理】 DNA是遗传信息的载体是最重要的生物信息分子是分子生物学研究的主要对象因此DNA的提取也应是分子生物学实验技术中最重要、最基本的操作。如不能有效的完成DNA提取方面的工作，那就根本谈不上进行分子生物学方面的实验。在DNA提取过程中应做到：1、根据不同研究需要，保证结构的相应完整性；2、尽量 ...

质粒DNA的提取是从事基因工程工作中的一项基本实验技术，但提取方法有很多种，以下介绍一种最常用的方法： 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、银染序列分析。方法如下： 1、接1%含质粒的大肠杆菌细胞于2ml LB培养基。 2、37℃振荡培养过夜。 3、取1.5ml菌体于Ep管，以4000rpm离心3min，弃上清液。 4、加0.lml溶液I(1％葡 ...

细菌基因组DNA的提取方法综述，提供了5种方法。 1 快速微量提取法A.取1.5ml菌体培养物于一灭菌Ep管中，12000rpm离心1min 丢去上清夜，收集菌体。B.加入400ul裂解液（40mMTris-醋酸，20mM醋酸钠1mMEDTA1%SDSpH7.8）混匀置于37oC水浴1hr。C.然后加入200ul5mol/L的氯化钠溶液，混匀后于13000rpm离心15min。D.取上清液，用苯酚 ...

一、目的 掌握植物基因组DNA提取的一般方法及注意事项。大分子量DNA分子的酶切分析。二、原理十六烷基三乙基溴化胺是一种去污剂，可溶解细胞膜，它能与核酸形成复合物，在高盐溶液中（0.7mol/L NaCl）是可溶的，当降低溶液盐浓度到一定程度(0.3 mol/L NaCl)时，从溶液中沉淀，通过离心就可将CTAB与核酸的复合物同蛋白质、多糖类物质分开，然后将CTAB与核酸的复合物沉淀溶解于高盐溶液 ...

DNA Extraction from Archival Formalin-fixed Paraffin-embedded Tissue SectionsAuthor: Shi et al. Source: Contributed by APostodoc Abstract: Describes two methods of extracting DNA from archived paraffi ...

TA CloningI. Initial mixture5 μl dd H2O1 μl PCR product1 μl ligation buffer2 μl TA vector (add second to last)1 μl ligase (add last)Incubate overnight at 15℃.II. Electroporation1. Chill ...

1.Grind in mortar and pestle or Waring blender with 5-7 volumes buffer A per g tissue. Use MCE at 350 l/L and if necessary with 5 ml 1 M DIECA/L. 2.Squeeze through cheesecloth two layers of Miracloth. ...

PurposeDNA extraction without phenol extraction and centrifugation.Procedure1. Lysis: the lysis buffer (usually 0.5ml) is added to the tissue or cell ( for a 75cm2 flask add 5ml directly to the cell). ...

This method is derived from a procedure developed by Toby Bradshaw and the Poplar Molecular Genetics Cooperative. We have tested the procedure with a variety of Populus species as well as tobacco and ...

Purpose: Purification of oligonucleotides (which have already been purified by reverse phase) can increase the efficiency of site directed mutagenesis from around 30% to ~75% or greater in some cases ...

PrincipleThe nucleic acids contained within the gel slice are electrophoresed out of the gel funneled down into the v-shaped channel and trapped within a high salt cushion resting at the bottom of the ...

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 brush ...

Materials:Silica Suspension:add 2 g of silica to 15 ml of H2Owash 3x by centifugation at 2000 x g for 2 minestimate vol of silica and resuspend in 2 vol H2OSilica Wash Solution:50 mM NaCl 10 mM Tris 7 ...

- make a 5µm section to do an evaluation of the % tumour cells- make 50X50µm sections for the DNA isolation- put sections in a falcon tube containing 10 ml of 1XSE- add 100µg/ml prot. K (endconcentrat ...