DNA基础知识

Mutagenesis is a fundamentally important DNA technology which seeks to change the base sequence of DNA and test its effect on gene or DNA function. The mutagenesis can be conducted in vivo (in studies ...

Southern Analysis Procedure (for analysis of genomic DNA or ordering of clones): 1) Prepare genomic DNA (ref.p.9.22 Maniatis) from cultured cells using one T75 flask (wash two times with PBS and then ...

Non-radioactive Probes I. Via random hexamers 1. Solutions:10X hexa nt miundefined: 500 mM Tris-Cl pH 7.2100 mM MgCl21 mM dithioerythritol (DTE)2 mg/ml BSA62.5 A260 units/ml (1.56 mg/ml) random hexanucleotid ...

RNA/Zeta Probe Dot Blotting protocol1)Make up RNA (up to 20μg)dissolved in sterile H2 OTE or 0.5% SDS to 500μl with ice-cold sterile 10mM NaOH1mM EDTA and apply it to Zeta Probe membraneheld in ...

The standard solution typically used for both pre-hybridisation and hybridisation is based on that given in Maniatis et al.(1982)with both Denhart's solution and heterologous DNA being replaced by hep ...

About 1/2 to 1 cm of tail should be cut from properly marked mice (using toe or ear clipping etc) about the age of 2-3 weeks. Place these tails into marked 1.5 ml Eppindorf tubes for processing; they ...

1. Depurination of DNA fragments: Wash gel in 0.25 M HCl 5' (small gels) 7' (large gels)2. Denaturation: Wash gel in (0.5 M NaOH 1.5 M NaCl) for 20' (small) to 30' (large).3. Neutralization: Wash in ( ...

Analys of Genomic DNA by Southern Hybridization (Southern Blot) Outline: Localization of particular sequences within genomic DNA is usually accomplished by the transfer techniques described by Souther ...

reagents: DNA for labeling (concentration c 150 ng/µl) modified nucleotides: Biotin-16-dUTP Digoxigenin-11-dUTP conc. 1nmol/µl (Boehringer Mannheim) dNTPs (regular nucleotides): dATP dCTP dGTP 0.5 mM ...

SolutionsProcedure Design complementary oligonucleotides with compatible half sites on the ends (I use BamHI and BglII). Dry down 300 ng of the two purified oligos together and resuspend in 9 m l Q wi ...

This protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA probes.Solutions10 mM dNTP StocksTh ...

Wear gloves throughout and work in radiation area. Monitor area before and after use.Mix the following in an eppendorf tube:1. 0.5 microgram oligonucleotide dissolved in H2O.2. 3 microliters 10x kinas ...

Protocol for Annealing OligonucleotidesOligo Name:Lot Number:Total nmol:Volume of Annealing Buffer added:Oligo Name:Lot Number:Total nmol:Volume of Annealing Buffer added:Annealing Buffer: 10mM Tris p ...

In the hood:96 well dish with bacteriatitertechmicrotubesglass pipetteRemove colonies from each well using the titertech and place them into the coverPipette up and down to thoroughly mix the colonies ...

DNaseI FootprintintSolutions10X Binding Buffer200 mM Tris 8.0 200 m l 1M Tris pH 8.0500 mM NaCl 100 m l 5M NaCl10 mM EDTA 20 m l 0.5 M EDTA pH 8.0680 m l Qstore at room temperatureDNaseI Dilution Buff ...

An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average every DNA molecule is cut onc ...

一、 目的及要求。1、 了解质粒作为载体在基因工程中的作用2、 熟记提取质粒的基本原理，学习提取过程和方法3、 为下一步实验提供高纯度的DNA 样品二、原理：1、 质粒（Plasmid）：独立于染色体以外的双链、闭合、环状DNA，可自我复制。为基因工程中的常用载体（Vector）2、 作为载体的质粒需具备以下特点：1） 能自主复制。2） 具有多个限制性内切酶的单一酶切位点，又称为多克隆位点（mul ...

7.1实验原理 核酸分子杂交是通过配对碱基对之间的非共价键（主要是氢键）结合，从而形成稳定的双链区。杂交分子的形成并不要求两条单链的碱基顺序完全互补，所以不同来源的核酸单链只要彼此之间有一定程度的互补顺序（即某种程度的同源性）就可以形成杂交双链。分子杂交可在DNA与DNA、RNA与RNA或RNA与DNA的二条单链之间进行。由于DNA一般都以双链形式存在，因此在进行分子杂交时，应先将双链DNA分子解 ...

一、直接检出病毒核酸 　　1．核酸杂交（Nucleic acid hybridization） -临床病毒学中报速诊断方法通常是检测标本中的病毒抗原，然而核酸分子杂交具有高度敏感性和特异性，斑点杂交 (Dot hybridization) 广泛用于检测呼吸道标本，尿标本中的病毒核酸。标本滴加到硝酸纤维素膜上，病毒DNA结合到膜上，在原位进行硷变性处理后，有放射标记的已知病毒DNA片段杂并，两条单股 ...

实验前的准备工作：一、实验器具与材料： 1、移液枪：1ml、200μl、20μl、10μl、2μl 2、吸头：1ml、200μl、20μl 3、匀浆管：5ml 4、吸头台：放置1ml吸头的一个，放置20μl吸头的一个 5、EP管：1.5ml、0.2ml、100μl 6、试剂瓶：2个60ml的棕色试剂瓶（广口，带盖） 1个125ml的白色试剂瓶（放无水乙醇） 7、量筒：50ml、250ml、500m ...