PartialEndonucleaseDigestion
Partial Endonuclease DigestionPrepare a 100 µl reaction mixture containing the DNA of interest in the appropriate 1X restriction enzyme buffer. Divide the reaction mixture between five prechilled microcentrifuge tubes such that tube 1 contains 30 µl, tubes 2 through 4 contain 20 µl, and tube 5 contains 10 µl (see diagram below). Place the tubes on ice.Add restriction endonuclease (3-10 units of enzyme per µg of DNA) ......阅读全文
Partial－Endonuclease－Digestion
Partial Endonuclease DigestionPrepare a 100 µl reaction mixture containing the DNA of interest in the appropriate 1X restriction enzyme buffer. Divide
DNA的酶学操作
DNA的酶学操作DNA Modifying Enzymes (Michael Blaber)Introduction to bacterial restriction/modification system. It provides very useful background knowledge
Construction－of－BAC－Libraries：Construction－of－a－BAC－library
Once high molecular weight (HMW) DNA has been prepared it must somehow be fragmented and DNA in the desired size range isolated. In general, as the de
Restriction－digestion
Restriction digestionRestriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction
Cyanogen－Bromide－digestion－of－protein
1. Proteins are TCA precipitated and washed with acetone, then dried.2. The CNBr should be brought to room temperature in the hood and used ONLY in th
Restriction－Enzyme－Digestion－of－DNA
Materials:10X restriction enzyme buffer (see manufacturer's recommendation)DNAsterile waterrestriction enzymephenol:chloroform (1:1)Add the follow
酶切反应(Setting－Up－a－Restriction－Endonuclease－Reaction)
一、 建立一个标准的酶切反应目前大多数研究者遵循一条规则，即10个单位的内切酶可以切割1μg不同来源和纯度的DNA。通常，一个50μl的反应体系中，1μl的酶在1X NEBuffer终浓度及相应温度条件下反应1小时即可降解1μg已纯化好的DNA。如果加入更多的酶，则可相应缩短反应时间；如果减少酶
Ingel－digestion－of－proteins－for－peptide－fingerprint－mapping
Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, the development of two-dimension
InGel－Digestion－of－Proteins－Separated－byPolyacrylamide－Gel－Electrophoresis
1. Excision of protein bands (spots) from polyacrylamide gelsRinse the gloves you use with water to avoid traces of dust in your sample.Rinse the gel
Genomic－Libraries
Genomic DNA libraries Size of some genomes and chromosomes:Comparative Sequence Sizes(Bases)(yeast chromosome 3)350 ThousandEscherichia coli (bacteriu
Restriction－Digests－of－High－Molecular－Weight－Yeast－DNA
Purpose:To perform restriction digests of YACs for mapping using rare cutting enzymes or more conventional restriction endonuclease digestion.Time req
“OneStep－Digestion”快速酶解试剂盒
快速酶解试剂盒——更快、更准确地进行蛋白质表征和研究轻松简单• 流程简单，一步完成样品处理• 变性还原烷基化酶解全流程只需半小时结果可靠• 降低样品处理过程引入的蛋白修饰• 提高蛋白酶解重现性• 增强数据可信度“One-Step Digestion”快速酶解试
Dephosphorylation－of－DNA
De-phosphorylation of DNAThe preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonu
PCR的下游应用
· Agarose Gel Electrophoresis of PCR Products (Robert H. Cruickshank)· Agarose Gel Electrophoresis of PCR Products (Immunology Resourc
PCR的下游应用
・ Agarose Gel Electrophoresis of PCR Products(Robert H. Cruickshank)・ Agarose Gel Electrophoresis of PCR Products(Immunology Resource)
Mitochondrial－DNA－Isolation－from－Somatic－Embryogenic－Cell－Cultures－of－Larix
Mitochondrial DNA is isolated by a modification of the methods described by Wilson and Chourey (1984) and Radetzky (1990). Cell cultures at four days
DNA分子的限制性内切酶（Restriction－enzyme－,－endonuclease）消...
限制性内切酶可特异地结合于一段被称为限制酶识别序列的DNA 序列位点上并在此切割双链DNA 。绝大多数限制性内切酶识别长度为4、5或6个核苷酸且呈二重对称的特异序列，切割位点相对于二重对称轴的位置因酶而异。一些酶恰在对称轴处同时切割 DNA 双链而产生带平端的DNA 片段，另一些酶则在对称轴
酵母人工染色体
· Easy YAC Preparation Method (Andrew Davies,Shaw lab)· Screening YAC libraries (Donis Keller Lab)This is a method for screening YAC l
重组DNA的分离、克隆与测序实验手册2
C. Restriction digestionRestriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restrict
DNA的限制性内切酶酶切(restriction－endonuclease，RE)分析
【原理】限制性内切酶(restrictionendonuclease，RE)是由细菌自己产生的能识别双链DNA分子中的特定碱基顺序，并以内切方式水解核酸中的磷酸二酯键的核酸水解酶。它可分为三种类型：Ⅰ、Ⅱ和Ⅲ型，Ⅱ型酶就是通常所指的RE，能识别双链DNA的特异顺序，并在这个顺序内进行切割。它是基因工
Double－Digestion(双酶切反应)时Universal－Buffer(通用缓冲液)的...
Double Digestion(双酶切反应)时Universal Buffer(通用缓冲液)的使用表■ 说明使用二种酶同时进行DNA切断反应 (Double Digestion) 时，为了节省反应时间，通常希望在同一反应体系内进行。TaKaRa采用Universal Buffer表示系统，
E.Z.N.A.®－Total－RNA－Midi－Kit－Protocol－DNase－I－digestion－Protocol
实验概要E.Z.N.A.® Total RNA Midiprep Kit provides a rapid and easy method for the isolation of up to 600 ug of total RNA from cultured eukaryotic cells,
Double－Digestion(双酶切反应)时Universal－Buffer(通用缓冲液)的...
说明使用二种酶同时进行DNA切断反应 (Double Digestion) 时，为了节省反应时间，通常希望在同一反应体系内进行。TaKaRa采用Universal Buffer表示系统，并对每种酶表示了在各Universal Buffer中的相对活性。尽管如此，在进行Double Dig
质粒DNA的限制性内切酶（restriction－endonuclease,－RE）酶切及..
实验原理： 限制性内切酶（restriction endonuclease, RE）能特异地结合于一段被称为限制性酶识别序列的DNA序列之内或其附近的特异位点上，并切割双链DNA。限制性内切酶可分为三类：I、Ⅱ和III类。I类和III类酶在同一蛋白质分子中兼有修饰（甲基
DNA－Isolation－From－BAC－－PAC－Clones
DNA Isolation From BAC & PAC Clones This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a
酵母准备
Yeast DNA PreparationYeast Genomic Preparation (Gottschling Lab)Rapid method for yeast genomic DNA isolation Yeast DNA Preparation (rapid glass bead
Construction－of－BAC－Libraries：Megabase－DNA－Isolation
Megabase DNA IsolationMegabase-size DNA isolation from plantsTo construct large insert DNA libraries in BAC and YAC vectors, methods must be developed
Basic－procedures－for－bacteria－culture1
A. Phenol extraction of DNA samplesPhenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated
酵母遗传学技术
Genome-wide Gene Expression Analysis (Richard Young Research Group,Whitehead Institute for Biomedical Research)A genoe-wide gene expression analysis u
General－Cloning－Protocols
Large Scale Preps: (See Large scale plsasmid prep protocol for more details)Cultures: Inoculate a 5 mL LB/Amp (50 - 100 µg/mL) culture in early a.m. w