Directe d Mutagenesis
|
|
- Corey Briggs
- 6 years ago
- Views:
Transcription
1 Directe d Mutagenesis A Practical Approac h M. J. McPHERSON
2
3 1. Mutagenesis facilitated by the removal or introduction of unique restriction sites 1 P. Carte r 1. Introduction to site-directed mutagenesis 1 Mutagenesis using mismatched oligonucleotides 1 Factors reducing yield 2 Strategies to enhance mutant yield 4 Phagemid vectors 5 Design of mutagenic oligonucleotide primers 6 Choice of endonuclease for restriction-selection and restriction - purification 8 Methods included in this chapter 9 2. Oligonucleotide-directed mutagenesis facilitated b y using repair deficient host strains and restrictionselection or restriction-purification 9 Enzymes 9 Other reagents 9 Bacterial and phage strains 1 0 Media 1 0 Preparation of single-stranded phagemid DNA 1 0 Construction of site-directed mutations Trouble-shooting procedures 1 9 Likely origin of clones other than target mutant 1 9 Checks on the efficiency of mutagenesis 20 Specificity of priming of mutagenic oligonucleotide 2 1 Purity of mutagenic oligonucleotide 2 1 Extent of phosphorylation of mutant oligonucleotide Advantages and limitations of restriction-selection and restriction-purification 2 2 Acknowledgements 2 3 References 23
4 2. Site-directed in vitro mutagenesis usin g uracil-containing DNA and phagemi d vectors 27 P. D. Yuckenberg, F. Witney, J. Geisselsoder, and J. McClary 1. Introduction Mutagenesis procedures 3 1 Bacteriology 3 1 Introduction of phagemid into E. coli CJ Isolation of uracil-containing phagemid particles 3 6 Extraction of phagemid DNA 3 8 Synthesis of the mutagenic strand 3 9 Use of T4 gene 32 protein 45 Gel analysis of the reaction products 46 Transformation of the reaction mixture 46 Analysis of transformants by DNA sequencing 47 References Phosphorothioate-based site-directed mutagenesis for single-stranded vectors 49 J. Sayers and F. Eckstei n 1. Introduction 49 Development of the methodology Mutagenesis procedure 50 Preparation of single-stranded template DNA 50 The mismatch oligonucleotide 5 4 Preparation of RF-IV DNA 5 5 Filtration through nitrocellulose 5 7 Nicking reaction 5 8 Gapping reaction 5 9 Preparation of the mutant homoduplex : the repolymerization step 62 Transfection Monitoring the procedure and debugging Modifications of the basic system Scope and limitations of the procedure 67 References 68
5 4. Site-directed mutagenesis using gappedheteroduplex plasmid DNA 7 1 S. Inouye and M. Inouye 1. Introduction The plasmid mutagenesis method 72 Preparation of plasmid DNA fragments I and II 72 Design and preparation of oligonucleotides 75 Denaturation and renaturation 76 Primer extension and transformation 77 Screening and confirmation of the mutations Concluding remarks 8 1 References Phosphorothioate-based double-strande d plasmid mutagenesis 83 D. B. Olsen and F. Eckstein 1. Introduction Overview of the methodology 83 Preparation of the double-stranded DNA templates 84 Preparation of mutant heteroduplex 88 Creation of the mutant homoduplex 93 Transformation of the mutant DNA Monitoring the procedure by agarose gel electrophoresis Discussion 98 Acknowledgements 98 References Linker scanning mutagenesis 10 1 B. Luckow and G. Schütz 1. Introduction 101 Definition of linker scanning mutagenesis 10 1 Different strategies for the construction of linker scannin g mutations 10 1 Alternative scanning mutagenesis procedures Construction of linker scanning mutants 104 Apurination of plasmid DNA by formic acid 104 Generation of nicks and small gaps in apurinic DNA b y exonuclease III 105 Linearization of nicked or gapped plasmid DNA b y nuclease SI 106
6 Ligation of linkers 10 8 Generation of DNA pool I 11 1 Generation of DNA pool II 11 5 Removal of the kanamycin resistance fragment 11 7 Size fractionation of mutated inserts 11 9 Identification of putative linker scanning mutations on th e basis of the topoisomer pattern General aspects of linker scanning mutagenesis 129 Selecting a suitable DNA fragment 129 Selecting a suitable vector 129 Selecting a suitable linker 130 Essential steps 130 Potential problems 13 0 Advantages 13 1 Time requirements 13 2 Acknowledgements 132 References Random chemical mutagenesis and th e non-selective isolation of mutated DNA sequences in vitro 13 5 C. Walton, R. K. Booth, and P. G. Stockley 1. Introduction Applications of random chemical mutagenesis 135 Random versus directed mutagenesis in vitro Chemistry of random mutagenesis in vitro 136 Background to chemical mutagenesis in vitro 136 Nitrous acid mutagenesis 137 Hydroxylamine mutagenesis 137 Bisulphite mutagenesis 137 Hydrazine mutagenesis 137 Acid depurination of DNA Practical considerations for random chemica l mutagenesis 13 8 Vector damage 13 8 Distribution of induced mutations 139 Considerations in the choice of target fragment(s) 139 Vectors for mutagenesis of single-stranded DNA 14 1 Mutagenic procedures 143 Regeneration of duplex DNA from treated single-strande d DNA 145 Amplification and enrichment of treated sequences 146 Nature of the mutations generated 148
7 5. Electrophoretic techniques allowing the enrichment o f mutants 14 9 Theory of denaturing gradient gel electrophoresis (DGGE) 150 Apparatus 15 1 Determination of optimum conditions for DGGE 15 3 Preparative denaturing gels 159 Analytical denaturing gradient gel electrophoresis 160 References An enzymatic method for the complet e mutagenesis of genes 163 J. K. C. Knowles and P. Lehtovaara 1. Introduction 163 Other random mutagenesis methods Principle of the method 164 Advantages of the method Practical aspects of the method 16 6 Isolation of template DNA 16 6 Base-specific limited elongation 16 7 Misincorporation and elongation 17 0 Transformation 17 2 Mutant characterization Conclusions 17 3 References Spiked oligonucleotide mutagenesis 17 7 S. C. Blacklow and J. R. Knowles 1. Introduction 177 Overview and theory 177 Strategy for spiked oligonucleotide-directed mutagenesis Materials 182 Buffers and solutions 182 Media 184 Bacterial strains Procedures 185 Preparation of oligonucleotides for mutagenesis 185 Preparation of single-stranded template (for mutagenesis) 189 Oligonucleotide-directed mutagenesis 192 Preparation of competent cells and transformation 192 Determination of mutagenesis efficiency by sequencing 194 Amplification of mutant plasmid DNA 196
8 4. Summary 19 8 Acknowledgements 19 8 References Cassette mutagenesis 19 9 J. H. Richards 1. Introduction Requirement for restriction sites Preparation of vector to receive a cassette 201 Restriction digest of plasmid DNA Preparation of cassette for insertion 203 Discrete sequence cassette 204 Multiple sequence cassette 205 Preparation of double-stranded cassettes Ligation Transformation 21 3 References Recombination and mutagenesis of DN A sequences using PCR 21 7 R. M. Horton and L. R. Pease 1. Introduction 21 7 The third generation of recombinant DNA technology 21 8 Synthetic applications of PCR Overlap extension 218 Mechanism 218 Mutagenesis 222 Recombination 222 What happens to the non-productive strands? Examples of overlap extension applications 226 Mutagenesis 226 Recombination Planning a strategy 228 Is PCR the solution to your problem? 228 Oligonucleotide design 23 1 Establishing a cassette system Procedures 236 Purifying the oligonucleotides 236 Determining the amount of primer to add 236
Recitation CHAPTER 9 DNA Technologies
Recitation CHAPTER 9 DNA Technologies DNA Cloning: General Scheme A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. (A single chromosome is shown
More informationVOLUME 2. Molecular Clonin g A LABORATORY MANUA L THIRD EDITIO N. Joseph Sambrook. David W. Russell
VOLUME 2 Molecular Clonin g A LABORATORY MANUA L THIRD EDITIO N Joseph Sambrook David W. Russell Chapter 8 In Vitro Amplification of DNA by the Polymerase 8. 1 Chain Reaction 1 The Basic Polymerase Chain
More informationLecture Four. Molecular Approaches I: Nucleic Acids
Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single
More informationGenetics and Genomics in Medicine Chapter 3. Questions & Answers
Genetics and Genomics in Medicine Chapter 3 Multiple Choice Questions Questions & Answers Question 3.1 Which of the following statements, if any, is false? a) Amplifying DNA means making many identical
More informationContents... vii. List of Figures... xii. List of Tables... xiv. Abbreviatons... xv. Summary... xvii. 1. Introduction In vitro evolution...
vii Contents Contents... vii List of Figures... xii List of Tables... xiv Abbreviatons... xv Summary... xvii 1. Introduction...1 1.1 In vitro evolution... 1 1.2 Phage Display Technology... 3 1.3 Cell surface
More informationBasics of Recombinant DNA Technology Biochemistry 302. March 5, 2004 Bob Kelm
Basics of Recombinant DNA Technology Biochemistry 302 March 5, 2004 Bob Kelm Applications of recombinant DNA technology Mapping and identifying genes (DNA cloning) Propagating genes (DNA subcloning) Modifying
More information3 Designing Primers for Site-Directed Mutagenesis
3 Designing Primers for Site-Directed Mutagenesis 3.1 Learning Objectives During the next two labs you will learn the basics of site-directed mutagenesis: you will design primers for the mutants you designed
More informationMolecular Biology Techniques Supporting IBBE
Molecular Biology Techniques Supporting IBBE Jared Cartwright Protein Production Lab Head Contact Details: email jared.cartwright@york.ac.uk Phone 01904 328797 Presentation Aims Gene synthesis Cloning
More informationBEST QUALITY HIGHEST PURITY. Recombinant ENZYMES & PROTEINS
BEST QUALITY HIGHEST PURITY Recombinant ENZYMES & PROTEINS We offer a wide range of highest quality enzymes and proteins for molecular biology including DNA polymerases, reverse transcriptases, DNA ligases,
More informationNZYGene Synthesis kit
Kit components Component Concentration Amount NZYGene Synthesis kit Catalogue number: MB33901, 10 reactions GS DNA Polymerase 1U/ μl 30 μl Reaction Buffer for GS DNA Polymerase 10 150 μl dntp mix 2 mm
More informationReading Lecture 8: Lecture 9: Lecture 8. DNA Libraries. Definition Types Construction
Lecture 8 Reading Lecture 8: 96-110 Lecture 9: 111-120 DNA Libraries Definition Types Construction 142 DNA Libraries A DNA library is a collection of clones of genomic fragments or cdnas from a certain
More informationAmplified segment of DNA can be purified from bacteria in sufficient quantity and quality for :
Transformation Insertion of DNA of interest Amplification Amplified segment of DNA can be purified from bacteria in sufficient quantity and quality for : DNA Sequence. Understand relatedness of genes and
More informationDesign. Construction. Characterization
Design Construction Characterization DNA mrna (messenger) A C C transcription translation C A C protein His A T G C T A C G Plasmids replicon copy number incompatibility selection marker origin of replication
More informationMolecular Cell Biology - Problem Drill 11: Recombinant DNA
Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna
More informationThe GeneEditor TM in vitro Mutagenesis System: Site- Directed Mutagenesis Using Altered Beta-Lactamase Specificity
Promega Notes Magazine Number 62, 1997, p. 02 The GeneEditor TM in vitro Mutagenesis System: Site- Directed Mutagenesis Using Altered Beta-Lactamase Specificity By Christine Andrews and Scott Lesley Promega
More informationData Sheet Quick PCR Cloning Kit
Data Sheet Quick PCR Cloning Kit 6044 Cornerstone Ct. West, Ste. E DESCRIPTION: The Quick PCR Cloning Kit is a simple and highly efficient method to insert any gene or DNA fragment into a vector, without
More informationBi 8 Lecture 4. Ellen Rothenberg 14 January Reading: from Alberts Ch. 8
Bi 8 Lecture 4 DNA approaches: How we know what we know Ellen Rothenberg 14 January 2016 Reading: from Alberts Ch. 8 Central concept: DNA or RNA polymer length as an identifying feature RNA has intrinsically
More informationMUTAGENESIS OF THE PROMOTER OF THE OIL PALM HOMOGENTISATE GERANYLGERANYL TRANSFERASE GENE (HGGT) BY PCR-DRIVEN OVERLAP EXTENSION
ICBAA2017-30 MUTAGENESIS OF THE PROMOTER OF THE OIL PALM HOMOGENTISATE GERANYLGERANYL TRANSFERASE GENE (HGGT) BY PCR-DRIVEN OVERLAP EXTENSION Mohd Shahrul Nizwanshah Karim and Siti Nor Akmar Abdullah Laboratory
More informationSome types of Mutagenesis
Mutagenesis What Is a Mutation? Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine
More informationSite-directed Mutagenesis
Site-directed Mutagenesis Applications Subtilisin (Met à Ala mutation resistant to oxidation) Fluorescent proteins Protein structure-function Substrate trapping mutants Identify regulatory regions/sequences
More informationDNA Ligation Kit Ver. 1 Manual
Table of content Description... 2 Procedures and Examples A. Insertion of DNA into plasmid vectors... 3 B. Insertion of DNA into λ phage vectors... 4 C. Self-circulization of linear DNA... 4 D. Linker
More informationDNA Technology. Asilomar Singer, Zinder, Brenner, Berg
DNA Technology Asilomar 1973. Singer, Zinder, Brenner, Berg DNA Technology The following are some of the most important molecular methods we will be using in this course. They will be used, among other
More informationHy-Fy High Fidelity Mix (x2)
Hy-Fy High Fidelity Mix (x2) #EZ-2021 1ml, 100rxn of 20μl Contents: 2X High Fidelity Mix 1ml Nuclease-free water 1ml Store at -20 C Shelf life: 2 years Description Hy-Fy High Fidelity Mix (x2) is a premixed,
More informationChapter 5. Objectives: Exploration of gene Recombinant DNA technology Genome sequencing Manipulation of Eukaryotic genes
Chapter 5 Objectives: Exploration of gene Recombinant DNA technology Genome sequencing Manipulation of Eukaryotic genes Restriction enzymes - cleave DNA et specific sequence - found in prokaryotes, cleave
More informationPLNT2530 (2018) Unit 6b Sequence Libraries
PLNT2530 (2018) Unit 6b Sequence Libraries Molecular Biotechnology (Ch 4) Analysis of Genes and Genomes (Ch 5) Unless otherwise cited or referenced, all content of this presenataion is licensed under the
More informationWeaver 4th ed Ch 4+5 Methods etc
Methods Weaver 4th ed Ch 4+5 Methods etc Chapter 4 Cloning pg 52 Restriction endonucleases pg 54 Vectors pg 56 Replica plating pg 63 Probes pg 64 cdnas pg 66 RACE (Rapid amplification of cdna ends) pg
More informationChapter 6 - Molecular Genetic Techniques
Chapter 6 - Molecular Genetic Techniques Two objects of molecular & genetic technologies For analysis For generation Molecular genetic technologies! For analysis DNA gel electrophoresis Southern blotting
More informationBacterial DNA replication
Bacterial DNA replication Summary: What problems do these proteins solve? Tyr OH attacks PO4 and forms a covalent intermediate Structural changes in the protein open the gap by 20 Å! 1 Summary: What problems
More informationCat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix
Molecular Cloning Laboratories User Manual Version 3.3 Product name: Choo-Choo Cloning Kits Cat #: CCK-10, CCK-20, CCK-096, CCK-384 Description: Choo-Choo Cloning is a highly efficient directional PCR
More informationContents. 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA Introduction Principle...
Contents 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA... 1 Introduction... 1 Principle... 1 Reagents Required and Their Role... 2 Procedure... 3 Observation... 4 Result
More informationManipulation of Purified DNA
Manipulation of Purified DNA To produce the recombinant DNA molecule, the vector, as well as the DNA to be cloned, must be cut at specific points and then joined together in a controlled manner by DNA
More information7.1 Techniques for Producing and Analyzing DNA. SBI4U Ms. Ho-Lau
7.1 Techniques for Producing and Analyzing DNA SBI4U Ms. Ho-Lau What is Biotechnology? From Merriam-Webster: the manipulation of living organisms or their components to produce useful usually commercial
More informationExam 2 Key - Spring 2008 A#: Please see us if you have any questions!
Page 1 of 5 Exam 2 Key - Spring 2008 A#: Please see us if you have any questions! 1. A mutation in which parts of two nonhomologous chromosomes change places is called a(n) A. translocation. B. transition.
More informationTechnical tips Session 4
Technical tips Session 4 Biotinylation assay: Streptavidin is a small bacterial protein that binds with high affinity to the vitamin biotin. This streptavidin-biotin combination can be used to link molecules
More informationBIOTECHNOLOGY : PRINCIPLES AND PROCESSES
CHAPTER 11 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES POINTS TO REMEMBER Bacteriophage : A virus that infects bacteria. Bioreactor : A large vessel in which raw materials are biologically converted into
More informationComputational Biology I LSM5191
Computational Biology I LSM5191 Lecture 5 Notes: Genetic manipulation & Molecular Biology techniques Broad Overview of: Enzymatic tools in Molecular Biology Gel electrophoresis Restriction mapping DNA
More informationBootcamp: Molecular Biology Techniques and Interpretation
Bootcamp: Molecular Biology Techniques and Interpretation Bi8 Winter 2016 Today s outline Detecting and quantifying nucleic acids and proteins: Basic nucleic acid properties Hybridization PCR and Designing
More informationMolecular Genetics Techniques. BIT 220 Chapter 20
Molecular Genetics Techniques BIT 220 Chapter 20 What is Cloning? Recombinant DNA technologies 1. Producing Recombinant DNA molecule Incorporate gene of interest into plasmid (cloning vector) 2. Recombinant
More informationChapter 8: Recombinant DNA. Ways this technology touches us. Overview. Genetic Engineering
Chapter 8 Recombinant DNA and Genetic Engineering Genetic manipulation Ways this technology touches us Criminal justice The Justice Project, started by law students to advocate for DNA testing of Death
More informationFast and efficient site-directed mutagenesis with Platinum SuperFi DNA Polymerase
APPLICATION NOTE Platinum Superi Polymerase ast and efficient site-directed mutagenesis with Platinum Superi Polymerase Introduction Site-directed mutagenesis is one of the most essential techniques to
More information1
1 2 3 4 5 Cosmids are plasmid vectors that contain cos sites. The cos site is the only requirement for DNA to be packaged into a phage particle 6 7 8 9 10 11 12 13 14 15 16 For de novo sequencing using
More informationIn vitro mutagenesis
Core course BMS361N Genetic Engineering In vitro mutagenesis Prof. Narkunaraja Shanmugam Dept. Of Biomedical Science School of Basic Medical Sciences Bharathidasan University In vitro mutagenesis Many
More information2014 Pearson Education, Inc. CH 8: Recombinant DNA Technology
CH 8: Recombinant DNA Technology Biotechnology the use of microorganisms to make practical products Recombinant DNA = DNA from 2 different sources What is Recombinant DNA Technology? modifying genomes
More information2054, Chap. 14, page 1
2054, Chap. 14, page 1 I. Recombinant DNA technology (Chapter 14) A. recombinant DNA technology = collection of methods used to perform genetic engineering 1. genetic engineering = deliberate modification
More informationLearning Objectives :
Learning Objectives : Understand the basic differences between genomic and cdna libraries Understand how genomic libraries are constructed Understand the purpose for having overlapping DNA fragments in
More informationMCB 102 University of California, Berkeley August 11 13, Problem Set 8
MCB 102 University of California, Berkeley August 11 13, 2009 Isabelle Philipp Handout Problem Set 8 The answer key will be posted by Tuesday August 11. Try to solve the problem sets always first without
More informationCH 8: Recombinant DNA Technology
CH 8: Recombinant DNA Technology Biotechnology the use of microorganisms to make practical products Recombinant DNA = DNA from 2 different sources What is Recombinant DNA Technology? modifying genomes
More informationHetero-Stagger PCR Cloning Kit
Product Name: Code No: Size: DynaExpress Hetero-Stagger PCR Cloning Kit DS150 20 reactions Kit Components: Box 1 (-20 ) phst-1 Vector, linearized Annealing Buffer Ligase Mixture phst Forward Sequence Primer
More informationSite directed mutagenesis, Insertional and Deletion Mutagenesis. Mitesh Shrestha
Site directed mutagenesis, Insertional and Deletion Mutagenesis Mitesh Shrestha Mutagenesis Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations
More informationMolecular Genetics II - Genetic Engineering Course (Supplementary notes)
1 von 12 21.02.2015 15:13 Molecular Genetics II - Genetic Engineering Course (Supplementary notes) Figures showing examples of cdna synthesis (currently 11 figures) cdna is a DNA copy synthesized from
More informationThe Biotechnology Toolbox
Chapter 15 The Biotechnology Toolbox Cutting and Pasting DNA Cutting DNA Restriction endonuclease or restriction enzymes Cellular protection mechanism for infected foreign DNA Recognition and cutting specific
More informationCharacteristics of bacterial Plasmid : Size : Conformation : Replication origin of replication : Replication Protein :
Characteristics of bacterial Plasmid : Size : Conformation : Replication origin of replication : Replication Protein : Definition of Plasmid Plasmids are extrachromosomal circular, double stranded DNA
More informationB. Incorrect! Ligation is also a necessary step for cloning.
Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of 10 1. Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease
More informationRecombinant DNA Technology
Recombinant DNA Technology Common General Cloning Strategy Target DNA from donor organism extracted, cut with restriction endonuclease and ligated into a cloning vector cut with compatible restriction
More informationCONTENTS. LIST OF FIGURES... i. LIST OF TABLES... iii. SUMMARY OF THE WORK DONE... vi
CONTENTS SECTION ~ LIST OF FIGURES... i LIST OF TABLES... iii LIST OF ABBREVIATIONS USED... ~... iv SUMMARY OF THE WORK DONE... vi 1. INTROD UCTION... :.................................... 1 1.1. AN OVERVIEW
More informationFast-Link DNA Ligation Kits
Fast-Link DNA Ligation Kits Cat. Nos. LK0750H and LK6201H Available exclusively thru Lucigen. lucigen.com/epibio www.lucigen.com MA086E Fast-Link DNA Ligation Kits 6/2017 1 1. Introduction The Fast-Link
More informationNxGen phi29 DNA Polymerase
NxGen phi29 DNA Polymerase Please read carefully and thoroughly before beginning FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll
More informationRecombinant DNA Technology
History of recombinant DNA technology Recombinant DNA Technology (DNA cloning) Majid Mojarrad Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists
More informationFun with DNA polymerase
Fun with DNA polymerase Why would we want to be able to make copies of DNA? Can you think of a situation where you have only a small amount and would like more? Enzymatic DNA synthesis To use DNA polymerase
More information4. Analysing genes II Isolate mutants*
.. 4. Analysing s II Isolate mutants* Using the mutant to isolate the classify mutants by complementation analysis wild type study phenotype of mutants mutant 1 - use mutant to isolate sequence put individual
More informationKOD -Plus- Mutagenesis Kit
Instruction manual KOD -Plus- Mutagenesis Kit 0811 F0936K KOD -Plus- Mutagenesis Kit SMK-101 20 reactions Store at -20 C Contents [1] Introduction [2] Flow chart [3] Components [4] Notes [5] Protocol 1.
More informationEnzymatic assembly of DNA molecules up to several hundred kilobases
nature methods Enzymatic assembly of DNA molecules up to several hundred kilobases Daniel G Gibson, Lei Young, Ray-Yuan Chuang, J Craig Venter, Clyde A Hutchison III & Hamilton O Smith Supplementary figures
More informationSELECTED TECHNIQUES AND APPLICATIONS IN MOLECULAR GENETICS
SELECTED TECHNIQUES APPLICATIONS IN MOLECULAR GENETICS Restriction Enzymes 15.1.1 The Discovery of Restriction Endonucleases p. 420 2 2, 3, 4, 6, 7, 8 Assigned Reading in Snustad 6th ed. 14.1.1 The Discovery
More informationFatchiyah
Fatchiyah Email: fatchiya@yahoo.co.id RNAs: mrna trna rrna RNAi DNAs: Protein: genome DNA cdna mikro-makro mono-poly single-multi Analysis: Identification human and animal disease Finger printing Sexing
More informationProblem Set 8. Answer Key
MCB 102 University of California, Berkeley August 11, 2009 Isabelle Philipp Online Document Problem Set 8 Answer Key 1. The Genetic Code (a) Are all amino acids encoded by the same number of codons? no
More informationNCERT. 2. An enzyme catalysing the removal of nucleotides from the ends of DNA is: a. endonuclease b. exonuclease c. DNA ligase d.
BIOTECHNOLOGY PRINCIPLES AND PROCESSES 75 CHAPTER 11 BIOTECHNOLOGY: PRINCIPLES AND PROCESSES 1. Rising of dough is due to: MULTIPLE-CHOICE QUESTIONS a. Multiplication of yeast b. Production of CO 2 c.
More informationGenBuilder TM Plus Cloning Kit User Manual
GenBuilder TM Plus Cloning Kit User Manual Cat. No. L00744 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2
More informationRecombinant DNA recombinant DNA DNA cloning gene cloning
DNA Technology Recombinant DNA In recombinant DNA, DNA from two different sources, often two species, are combined into the same DNA molecule. DNA cloning permits production of multiple copies of a specific
More informationGenBuilder TM Plus Cloning Kit User Manual
GenBuilder TM Plus Cloning Kit User Manual Cat.no L00744 Version 11242017 Ⅰ. Introduction... 2 I.1 Product Information... 2 I.2 Kit Contents and Storage... 2 I.3 GenBuilder Cloning Kit Workflow... 2 Ⅱ.
More informationIn Vitro DNA Recombination by Random Priming
DNA Recombination by Random Priming 99 13 In Vitro DNA Recombination by Random Priming Olga Esteban, Ryan D. Woodyer, and Huimin Zhao 1. Introduction Variation coupled to selection is the hallmark of natural
More informationSite-directed mutagenesis of proteins
IFM/Kemi Linköpings Universitet August 2013/LGM Labmanual Site-directed mutagenesis of proteins Figur 1: Flow-chart of the site-directed mutagenesis lab exercise 2 Site-specific mutagenesis Introduction
More informationXXII DNA cloning and sequencing. Outline
XXII DNA cloning and sequencing 1) Deriving DNA for cloning Outline 2) Vectors; forming recombinant DNA; cloning DNA; and screening for clones containing recombinant DNA [replica plating and autoradiography;
More informationCalifornia Institute of Technology. Directed evolution. Dr. F.H. Arnold s lab
Directed evolution. Dr. F.H. Arnold s lab May 4, 1999 Mutagenic PCR -[Mn] The amount of Mn used in the reaction should be titrated to produce the desired mutagenic rate. Libraries that have close to 30%
More informationChapter 9 Genetic Engineering
Chapter 9 Genetic Engineering Biotechnology: use of microbes to make a protein product Recombinant DNA Technology: Insertion or modification of genes to produce desired proteins Genetic engineering: manipulation
More informationFunctional Genomics Research Stream. Research Meetings: November 2 & 3, 2009 Next Generation Sequencing
Functional Genomics Research Stream Research Meetings: November 2 & 3, 2009 Next Generation Sequencing Current Issues Research Meetings: Meet with me this Thursday or Friday. (bring laboratory notebook
More informationMutagenesis PCR I (Multiple Site Directed Mutagenesis)
Mutagenesis Mutagenesis PCR I (Multiple Site Directed Mutagenesis) Mixture 25µl total reaction volume : 1. 2.5 µl of 10X Taq lligase buffer (need the NAD for Taq ligase) 2. 0.5 µl 100mM ATP 3. X µl (50-100
More informationCONSTRUCTION OF GENOMIC LIBRARY
MODULE 4-LECTURE 4 CONSTRUCTION OF GENOMIC LIBRARY 4-4.1. Introduction A genomic library is an organism specific collection of DNA covering the entire genome of an organism. It contains all DNA sequences
More informationEfficient Multi-site-directed Mutagenesis directly from Genomic Template.
Efficient Multi-site-directed Mutagenesis directly from Genomic Template. Fengtao Luo 1, Xiaolan Du 1, Tujun Weng 1, Xuan Wen 1, Junlan Huang 1, Lin Chen 1 Running title: Multi-site-directed Mutagenesis
More informationPlease sign below if you wish to have your grades posted by the last five digits of your SSN
BIO 226R EXAM III (Sample) PRINT YOUR NAME Please sign below if you wish to have your grades posted by the last five digits of your Signature BIO 226R Exam III has 8 pages, and 26 questions. There are
More informationGENETIC ENGINEERING worksheet
Section A: Genetic Engineering Overview 1. What is genetic engineering? 2. Put the steps of genetic engineering in order. Recombinant product is isolated, purified and analyzed before marketing. The DNA
More informationTable of Contents. i. Insertion of DNA fragments into plasmid vector...4. ii. Self-circularization of linear blunt-ended DNA...4
Table of Contents I. Description...2 II. Kit Components...2 III. Storage...2 IIV. Notes...2 V. Reference...3 VI. PROCEDURES A. Dephosphorylation of vector DNA...3 B. Blunting reaction...3 C. Ligation reaction
More informationp Kinesin light chain Kinesin heavy chain Kinesin heavy chain Kinesin light chain + - BCMA
p.959-970 Kinesin light chain Kinesin heavy chain Kinesin heavy chain Kinesin light chain + - BCMA - 2011-05 1 tetratricopeptide repeat Zona di possibile interazione con cargo BCMA - 2011-05 2 Verhey et
More informationAmplicons, Heteroduplexes and Enzymes - Proper Processing Elevates Detection of CRISPR Gene Editing Events
Amplicons, Heteroduplexes and Enzymes - Proper Processing Elevates Detection of CRISPR Gene Editing Events Steve Siembieda, MS MBA VP Commercialization ABRF Conference February 2015 What Is CRISPR? Clustered
More informationBIOLOGY - CLUTCH CH.20 - BIOTECHNOLOGY.
!! www.clutchprep.com CONCEPT: DNA CLONING DNA cloning is a technique that inserts a foreign gene into a living host to replicate the gene and produce gene products. Transformation the process by which
More informationHighly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase
Highly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase H. Liu, R. Ye and Y.Y. Wang Department of Medical Microbiology and Parasitology, School of
More informationQ5 Site-Directed Mutagenesis Kit
DNA MODIFYING ENZYMES Q5 Site-Directed Mutagenesis Kit Instruction Manual NEB #E0554S 10 reactions Version 1.0 1/13 be INSPIRED drive DISCOVERY stay GENUINE This product is intended for research purposes
More informationNEBNext RNase III RNA Fragmentation Module
SAMPLE PREPARATION NEBNext RNase III RNA Fragmentation Module Instruction Manual NEB #E6146S 100 reactions NEBNext RNase III RNA Fragmentation Module Table of Contents: Description....2 Applications....2
More informationSimple protocol for gene editing using GenCrisprTM Cas9 nuclease
Simple protocol for gene editing using GenCrisprTM Cas9 nuclease Contents Protocol Step 1: Choose the target DNA sequence Step 2: Design sgrna Step 3: Preparation for sgrna 3.1 In vitro transcription of
More informationAntisense RNA Targeting the First Periplasmic Domain of YidC in Escherichia coli Appears to Induce Filamentation but Does Not Affect Cell Viability
Antisense RNA Targeting the First Periplasmic Domain of YidC in Escherichia coli Appears to Induce Filamentation but Does Not Affect Cell Viability Riaaz Lalani, Nathaniel Susilo, Elisa Xiao, Andrea Xu
More information3. Translation. 2. Transcription. 1. Replication. and functioning through their expression in. Genes are units perpetuating themselves
Central Dogma Genes are units perpetuating themselves and functioning through their expression in the form of proteins 1 DNA RNA Protein 2 3 1. Replication 2. Transcription 3. Translation Spring 2002 21
More informationCRISPR/Cas9 Genome Editing: Transfection Methods
CRISPR/ Genome Editing: Transfection Methods For over 20 years Mirus Bio has developed and manufactured high performance transfection products and technologies. That expertise is now being applied to the
More informationPuro. Knockout Detection (KOD) Kit
Puro Knockout Detection (KOD) Kit Cat. No. CC-03 18 Oct. 2016 Contents I. Kit Contents and Storage II. Product Overview III. Methods Experimental Outline Genomic DNA Preparation Obtain Hybrid DNA Digest
More informationRequirements for the Genetic Material
Requirements for the Genetic Material 1. Replication Reproduced and transmitted faithfully from cell to cell-generation to generation. 2. Information Storage Biologically useful information in a stable
More informationXactEdit Cas9 Nuclease with NLS User Manual
XactEdit Cas9 Nuclease with NLS User Manual An RNA-guided recombinant endonuclease for efficient targeted DNA cleavage Catalog Numbers CE1000-50K, CE1000-50, CE1000-250, CE1001-250, CE1001-1000 Table of
More informationPrimeSTAR Max DNA Polymerase
Cat. # R045A For Research Use PrimeSTAR Max DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 General Composition of PCR Reaction Mixture...3 V.
More informationCRISPR/Cas9 Gene Editing Tools
CRISPR/Cas9 Gene Editing Tools - Guide-it Products for Successful CRISPR/Cas9 Gene Editing - Why choose Guide-it products? Optimized methods designed for speed and ease of use Complete kits that don t
More informationCHAPTER 9 DNA Technologies
CHAPTER 9 DNA Technologies Recombinant DNA Artificially created DNA that combines sequences that do not occur together in the nature Basis of much of the modern molecular biology Molecular cloning of genes
More informationChapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc.
Chapter 20 Recombinant DNA Technology Copyright 2009 Pearson Education, Inc. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors Recombinant DNA refers
More informationTable of Contents. I. Description...2. Components...2. Storage...2. Features...2. V. General Composition of PCR Reaction Mixture...
Table of Contents I. Description...2 II. Components...2 III. Storage...2 IV. Features...2 V. General Composition of PCR Reaction Mixture...5 VI. PCR Conditions...5 VII. Optimization of Parameters...6 VIII.
More informationFast-Link DNA Ligation Kits
Cat. Nos. LK11025, LK0750H, and LK6201H The provide reagents optimized for the construction of recombinant vectors in a short time. Ligation reactions require incubation for as little as 5 minutes at room
More information