Lecture 5 - Bacteriophage Vectors; Lab Practicum 2 (AMG text pp ) September 4, 2001
|
|
- Owen Conley
- 7 years ago
- Views:
Transcription
1 Lecture 5 - Bacteriophage Vectors; Lab Practicum 2 (AMG text pp ) September 4, 2001 Biology of Bacteriophage Lamda Lambda phage is a bacterial virus that infects E. coli, and depending on early events (and genetics), can either multiply within cells leading to cell lysis, or the viral DNA can integrate into the bacterial genome in a process called lysogeny. If lambda gene transcription is activated, then lytic infection occurs leading to phage release and infection of nearby E. coli cells. Alternatively, if lambda gene transcription is repressed, then the infection is lysogenic resulting in lambda DNA integration into the E. coli genome. Applied Molecular Genetic methods used in the lab are designed to take advantage of cell lysis. The plaque formation assay can be used to isolate pure strains of lambda phage by using a low multiplicity of infection (MOI). Bacteriophage plaques are physical areas on a petri plate where the bacteria have all lysed due to multiple rounds of infection by a single clonal phage. Each plaque contains about 1 million virus particles derived from a single infection event - the viral particles in a single plaque are therefore clonally related (same DNA sequence). Sometimes it is desireable to purify large amounts of phage for the purpose of isolating recombinant DNA from a clonal isolate. One method to do this is to infect a liquid culture of E. coli with phage stock and then harvest the lysate 4-8 hours later. The bacterial debris (lysed cells) is removed by low speed centrifugation and then polyethylene glycol (PEG) is added to the supernatent and the phage are then pelleted by high speed ultracentrifugation. What does it mean to "titer a phage stock" and how is it done? Why are the host bacteria grown in media containing the sugar maltose prior to infection? Why does it matter how much phage stock you add to the bacterial liquid culture, doesn't it all just mix around and lead to 100% infection eventually? What is the biochemical explanation for adding PEG to the phage suspension? Once you had obtained a highly concentrated stock of purified phage, how would you separate the recombinant phage DNA away from the phage head and tail proteins? Two important technical hurdles had to be overcome before lambda vectors could be utilized as cloning reagents for genomic and cdna libraries; 1) In vitro lambda phage packaging extracts were developed and made available commercially, 2) Genetic strategies were developed to increase the yield of recombinant phage in DNA libraries to make up for the low ratio of insert to vector DNA used in the initial ligation reactions. Page 1
2 Two such genetic strategies are shown below. The first was developed for cdna libraries and the second for genomic DNA libraries. Note that the total length of lambda DNA that can be efficiently packaged is a crucial determinant in this protein-driven process with a maximum limit of 52 kb (vector + insert). Insertional cloning into the ci gene of the lambda-gt10 cdna cloning vector (DNA inserts of ~1-5 kb) can be selected in hfl (high frequency of lysogeny) mutant strains of E. coli. In hfla strains of E. coli, expression of the lambda cii gene is elevated, resulting in transcriptional induction of the lambda ci repressor gene which promotes lysogeny. Disruption of the lambda ci coding sequence by DNA insertion into the unique EcoRI site of the lambda gt10 cdna cloning vector, blocks the lysogenic pathway leading to cell lysis and plaque formation. Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3 genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb, permits lytic growth of recombinant phage in E. coli strains containing the P2 bacteriophage lysogen. Does lambda DNA packaging require any enzymes or ATP hydrolysis? Why is it important to use low ratios of insert DNA to vector DNA in the construction of lambda DNA libraries, wouldn't it just be easier to increase the ratio to decrease the number of "empty vectors" in the library stock? M13 Bacteriophage Biology M13 filamentous phage have a single strand genome that exists temporarily inside infected E.coli cells as a double strand plasmid. M13 phage are budded off of an infected cell and single strand DNA can be purified for use in DNA sequencing or in vitro mutagenesis. Initially M13 phage vectors required a working knowledge of phage biology and was primarily used for creating single strand DNA molecules for DNA sequencing. Fortunately, M13-derived cloning vectors called "phagemids" have been developed which take advantage of M13 replication to produce single strand molecules, but can be propagated as conventional ColE1-based replicating double strand plasmids. Bluescript KS+ is an example of a phagemid cloning vector. Often times, phagemid vectors are used to prepare single strand DNA for in vitro mutagenesis protocols using oligonucleotide primers (chapter 3). Infection of E. coli F+ cells containing phagemid DNA with replication-deficient M13 helper phage results in the packaging of single strand phagemid DNA. The orientation of the M13 replication origin (+ or -), relative to the insert DNA, in the phagemid vector, determines which strand of the insert DNA (coding or non-coding) will be contained in the packaged phage. Since the bacterial sex pilus proteins are encoded on the F plasmid, and the pilus is required for M13 phage attachment, E. coli strains have been developed that include a transposon encoded antibiotic resistance gene on the F plasmid (tetr or kanr). Rolling Page 2
3 circle replication and phage packaging by helper virus proteins result in the production of recombinant M13 phage. Why is a helper phage required to produce single strand DNA from an M13 phagemid? What does the orientation-dependence of the M13 ori tell you about filamentous phage replication and how is this different than lambda phage replication? Lab Practicum 2 - Protein expression in E. coli using the pet system. High level expression of recombinant proteins in bacteria is a common process in Biotechnology. The production of human insulin in bacteria is one such example. Another example is the production of plant calreticulin protein in bacteria as a means to generate anti-calreticulin antibodies in rabbits. Probably the most widely used bacterial protein expression is the pet vector system which was developed in 1984 by Bill Studier and colleagues at Brookhaven National Labs. The vector system is now sold commercially by Novagen. One of the most innovative pet vectors is petblue which utilizes a lacz gene (alpha region) on the opposite coding strand such that blue-white screening can be done but the recombinant protein does not contain any lacz fusion sequences. Research Objective A plant biologist is interested in studying the calcium binding protein calreticulin in the plant Arabidopsis thaliana to determine its possible function in floral tissues. As a first step, he wants to generate antibodies that specifically recognize Arabidopsis calreticulins in order to determine which floral cell types express these proteins using immunocytochemistry. His plan is to express an Arabidopsis calreticulin coding sequence in E. coli to allow isolation of calreticulin antigen for polyclonal antibody production in rabbits. He searched the Arabidopsis expressed sequence tag (EST) database over the internet using a known human calreticulin sequence and identified several candidate cdna clones that were available from the Arabidopsis Biological Resource Center at Ohio State University. Available Information and Reagents 1. A 1.4 kb Arabidopsis cdna has been obtained from the Resource Center and its complete DNA sequence was determined. It was found to encode a 425 amino acid protein with high amino acid homology to human calreticulin. 2. A pet(his)6 bacterial expression vector, the plyss plasmid encoding T7 lysozyme, and the BL21(DE3) E. coli lde3 lysogen strain containing the T7 RNA polymerase gene under lac control, have been obtained from a commercial source. 3. Biochemical supplies are available for purifying proteins containing a polyhistidine sequence from bacterial cell extracts using metal chelation chromatography. Page 3
4 4. Resources are available for polyclonal antibody production in rabbits using purified antigen. The plant biologist has expertise in plant immunocytochemistry and biochemistry. Basic Strategy The two essential components of this T7 bacteriophage RNA polymerase-based expression system are the pet plasmid containing the coding sequence of a DNA insert downstream of the T7 promoter (Tpr), and an E. coli strain containing a genomic copy of the T7 RNA polymerase (T7 pol) gene under the control of the lac UV5 promoter Also shown in this example is a T7 lysozyme encoding plasmid containing the chloramphenicol-resistance gene (cat), which provides a means to inhibit the small amount of T7 RNA polymerase that is expressed in the absence of IPTG. High level expression of the histidine-tagged calreticulin protein is obtained by blocking laci repressor function with the addition of IPTG to the media. This IPTG-inducible expression system is based on the finding that bacteriophage T7 RNA polymerase is highly specific, and efficient, in transcribing genes containing a T7 promoter. By transforming a recombinant calreticulin pet vector into an E. coli strain containing the T7 RNA polymerase gene under lac control, it will be possible to induce calreticulin expression by treating the cells with IPTG (see below). Since low level expression of the T7 RNA polymerase gene occurs in this system in the absence of IPTG, and appreciable levels of the plant calreticulin protein may inhibit bacterial growth, a compatible plasmid containing the T7 lysozyme gene will also be introduced into the host cells. T7 lysozyme is a bifunctional protein that not only cleaves peptidoglycan linkages in the bacterial cell wall but also stoichiometrically inhibits the function of T7 RNA polymerase. Therefore, low level expression of T7 lysozyme is able to inhibit the activity of T7 RNA polymerase molecules present in cells even before IPTG treatment. Regulated expression of genes in E. coli using the pet vector system. The two essential components of this T7 bacteriophage RNA polymerase-based expression system are the pet plasmid containing the coding sequence of a DNA insert downstream of the T7 promoter (Tpr), and an E. coli strain containing a genomic copy of the T7 RNA polymerase (T7 pol) gene under the control of the lac UV5 promoter. Also shown in this example is a T7 lysozyme encoding plasmid containing the chloramphenicol-resistance gene (cat), which provides a means to inhibit the small amount of T7 RNA polymerase that is expressed in the absence of IPTG. High level expression of the histidine-tagged calreticulin protein is obtained by blocking laci repressor function with the addition of IPTG to the media. Flow scheme for calreticulin antibody production using purified protein from bacterial extracts. The polyhistidine tag contained within the recombinant calreticulin protein can be used to purify soluble protein from cell extracts using a Ni2+ affinity column. The bound calreticulin protein is eluted from the column with imidazole which outcompetes the histidines for Ni2+ binding. Protein purification is monitored by gel electrophoresis (SDS-PAGE). In this example, calreticulin-specific polyclonal antibodies are produced in a rabbit and immunopurified with a calreticulin affinity column that is Page 4
5 made using the calreticulin antigen protein from the E. coli extracts. The immunopurified calreticulin-specific antibodies could be useful as reagents in Western blots, immunocytochemical studies and immunoprecipitation experiments. Comments The production of antigen in E. coli is the least demanding application of bacterial expression systems since denaturing conditions can be used to aid in protein solubilization. The production of polyclonal antibodies using recombinant proteins has become a standard procedure. Many researchers use commercial services that specialize in antibody production. Protein samples are sent to the service by express mail, and 7-10 weeks later, serum is sent back for analysis. Since the antigen is produced in E. coli and therefore available in large quantities, antigen-specific antibodies can be purified from the serum using affinity chromatography. Depending on the results obtained using polyclonal antibodies, the production of antigen-specific monoclonal antibodies may also be warranted. Prospective The plant biologist found that the anti-calreticulin antibodies detected high levels of the protein in developing seeds, a result which is consistent with its role as a chaperone protein in tissues with elevated rates of protein synthesis. During seed development, large amounts of storage proteins are synthesized which are later degraded to fuel seed germination. It would be interesting to determine if calreticulin was required for seed development. This could be done for example, by disrupting calreticulin function using cell-specific antisense genetics or RNA interference (RNAi) and by characterizing calreticulin in Arabidopsis mutants containing defects in seed development. Calreticulin function in the seeds could also be studied by isolating seed proteins that interact with the bacterial expressed calreticulin (His)6 protein following absorption to a Ni2+ affinity column. What are two advantages of using the T7 promoter and T7 RNA polymerase in the pet system to control expression of cloned recombinant genes. How is this better than simply using the lac promoter upstream of the calreticulin gene)? What is the purpose of running the rabbit polyclonal anti-serum over a calreticulin affinity column? How are the antibodies eluted from the column without releasing the calreticulin antigen at the same time? Describe how the bacterially-expressed His-calreticulin protein could be used to isolate calreticulin bind proteins from seed extracts? How could you identify the bound proteins beyond their molecular weight on a gel? Page 5
restriction enzymes 350 Home R. Ward: Spring 2001
restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually
More informationDNA CLONING. DNA segment has been developed: polymerase chain reaction PCR. Viral DNA-s bacteriophage λ, filamentous bacteriophages
DNA CLONING - What is cloning? The isolation of discrete pieces of DNA from their host organism and their amplification through propagation in the same or a different host More recently an alternitive,
More informationExpression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu
Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.
More informationIntegrated Protein Services
Integrated Protein Services Custom protein expression & purification Version DC04-0012 Expression strategy The first step in the recombinant protein generation process is to design an appropriate expression
More informationMolecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION
Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN
More informationSTUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS
STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS THESIS SUBMITTED FOR THE DEGREB OF DOCTOR OF PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF CALCUTTA 1996 NRISINHA DE, M.Sc DEPARTMENT OF BIOCHEMISTRY
More informationHCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise:
HCS604.03 Exercise 1 Dr. Jones Spring 2005 Recombinant DNA (Molecular Cloning) exercise: The purpose of this exercise is to learn techniques used to create recombinant DNA or clone genes. You will clone
More informationBiotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College
Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology
More informationBacterial Transformation and Plasmid Purification. Chapter 5: Background
Bacterial Transformation and Plasmid Purification Chapter 5: Background History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment
More informationIntegrated Protein Services
Integrated Protein Services Custom protein expression & purification Last date of revision June 2015 Version DC04-0013 www.iba-lifesciences.com Expression strategy The first step in the recombinant protein
More informationRecombinant DNA and Biotechnology
Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study
More informationTransfection-Transfer of non-viral genetic material into eukaryotic cells. Infection/ Transduction- Transfer of viral genetic material into cells.
Transfection Key words: Transient transfection, Stable transfection, transfection methods, vector, plasmid, origin of replication, reporter gene/ protein, cloning site, promoter and enhancer, signal peptide,
More informationKMS-Specialist & Customized Biosimilar Service
KMS-Specialist & Customized Biosimilar Service 1. Polyclonal Antibody Development Service KMS offering a variety of Polyclonal Antibody Services to fit your research and production needs. we develop polyclonal
More informationGene Cloning. Reference. T.A. Brown, Gene Cloning, Chapman and Hall. S.B. Primrose, Molecular Biotechnology, Blackwell
Gene Cloning 2004 Seungwook Kim Chem. & Bio. Eng. Reference T.A. Brown, Gene Cloning, Chapman and Hall S.B. Primrose, Molecular Biotechnology, Blackwell Why Gene Cloning is Important? A century ago, Gregor
More information50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert
More informationRecombinant DNA Unit Exam
Recombinant DNA Unit Exam Question 1 Restriction enzymes are extensively used in molecular biology. Below are the recognition sites of two of these enzymes, BamHI and BclI. a) BamHI, cleaves after the
More informationRecombinant DNA Technology
Recombinant DNA Technology Dates in the Development of Gene Cloning: 1965 - plasmids 1967 - ligase 1970 - restriction endonucleases 1972 - first experiments in gene splicing 1974 - worldwide moratorium
More informationViral Infection: Receptors
Viral Infection: Receptors Receptors: Identification of receptors has come from expressing the gene for the receptor in a cell to which a virus does not normally bind -OR- By blocking virus attachment
More informationGENE CLONING AND RECOMBINANT DNA TECHNOLOGY
GENE CLONING AND RECOMBINANT DNA TECHNOLOGY What is recombinant DNA? DNA from 2 different sources (often from 2 different species) are combined together in vitro. Recombinant DNA forms the basis of cloning.
More informationCHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA
CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the
More informationRecombinant DNA & Genetic Engineering. Tools for Genetic Manipulation
Recombinant DNA & Genetic Engineering g Genetic Manipulation: Tools Kathleen Hill Associate Professor Department of Biology The University of Western Ontario Tools for Genetic Manipulation DNA, RNA, cdna
More informationLab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity
Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency
More informationBiotechnology: DNA Technology & Genomics
Chapter 20. Biotechnology: DNA Technology & Genomics 2003-2004 The BIG Questions How can we use our knowledge of DNA to: diagnose disease or defect? cure disease or defect? change/improve organisms? What
More informationBacterial and Phage Genetic Switches
Bacterial and Phage Genetic Switches Prof. C. J. Dorman Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin. Lecture 1 The genetic switch controlling the lytic-lysogen
More informationChapter 18: Applications of Immunology
Chapter 18: Applications of Immunology 1. Vaccinations 2. Monoclonal vs Polyclonal Ab 3. Diagnostic Immunology 1. Vaccinations What is Vaccination? A method of inducing artificial immunity by exposing
More informationProtein Expression and Analysis. Vijay Yajnik, MD, PhD GI Unit MGH
Protein Expression and Analysis Vijay Yajnik, MD, PhD GI Unit MGH Identify your needs Antigen production Biochemical studies Cell Biology Protein interaction studies including proteomics Structural studies
More informationCUSTOM ANTIBODIES. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation
CUSTOM ANTIBODIES Highly competitive pricing without compromising quality. Rat monoclonal antibodies for the study of gene expression and proteomics in mice and in mouse models of human diseases available.
More information4. DNA replication Pages: 979-984 Difficulty: 2 Ans: C Which one of the following statements about enzymes that interact with DNA is true?
Chapter 25 DNA Metabolism Multiple Choice Questions 1. DNA replication Page: 977 Difficulty: 2 Ans: C The Meselson-Stahl experiment established that: A) DNA polymerase has a crucial role in DNA synthesis.
More informationINTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B
INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE QUALITY OF BIOTECHNOLOGICAL PRODUCTS: ANALYSIS
More informationHow To Understand How Gene Expression Is Regulated
What makes cells different from each other? How do cells respond to information from environment? Regulation of: - Transcription - prokaryotes - eukaryotes - mrna splicing - mrna localisation and translation
More informationMilestones of bacterial genetic research:
Milestones of bacterial genetic research: 1944 Avery's pneumococcal transformation experiment shows that DNA is the hereditary material 1946 Lederberg & Tatum describes bacterial conjugation using biochemical
More informationStructure and Function of DNA
Structure and Function of DNA DNA and RNA Structure DNA and RNA are nucleic acids. They consist of chemical units called nucleotides. The nucleotides are joined by a sugar-phosphate backbone. The four
More informationFirst Strand cdna Synthesis
380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences
More information2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line
i 1 INTRODUCTION 1.1 Human Hepatitis B virus (HBV) 1 1.1.1 Pathogenesis of Hepatitis B 1 1.1.2 Genome organization of HBV 3 1.1.3 Structure of HBV virion 5 1.1.4 HBV life cycle 5 1.1.5 Experimental models
More informationfrom Cloned Genes Learning outcomes: By the end of this chapter you will have an understanding of:
9 Production of Proteins from Cloned Genes Learning outcomes: By the end of this chapter you will have an understanding of: the reasons for producing proteins from cloned genes some of the more common
More informationModule 3 Questions. 7. Chemotaxis is an example of signal transduction. Explain, with the use of diagrams.
Module 3 Questions Section 1. Essay and Short Answers. Use diagrams wherever possible 1. With the use of a diagram, provide an overview of the general regulation strategies available to a bacterial cell.
More informationDNA Fingerprinting. Unless they are identical twins, individuals have unique DNA
DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence
More informationCCR Biology - Chapter 9 Practice Test - Summer 2012
Name: Class: Date: CCR Biology - Chapter 9 Practice Test - Summer 2012 Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Genetic engineering is possible
More informationBio-Reagents Gene synthesis Peptide Synthesis Protein Expression Antibody Production. Life Science Products and Services
Bio-Reagents Gene synthesis Peptide Synthesis Protein Expression Antibody Production Life Science Products and Services Since 2002, Biomatik has provided worldwide researchers in life science discovery
More informationApplication Guide... 2
Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied
More informationGenetics Test Biology I
Genetics Test Biology I Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Avery s experiments showed that bacteria are transformed by a. RNA. c. proteins.
More informationProtein Expression. A Practical Approach J. HIGGIN S
Protein Expression A Practical Approach S. J. HIGGIN S B. D. HAMES List of contributors Abbreviations xv Xvi i 1. Protein expression in mammalian cell s Marlies Otter-Nilsson and Tommy Nilsso n 1. Introduction
More informationBCOR101 Midterm II Wednesday, October 26, 2005
BCOR101 Midterm II Wednesday, October 26, 2005 Name Key Please show all of your work. 1. A donor strain is trp+, pro+, met+ and a recipient strain is trp-, pro-, met-. The donor strain is infected with
More informationHighPure Maxi Plasmid Kit
HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer
More informationGenetic Technology. Name: Class: Date: Multiple Choice Identify the choice that best completes the statement or answers the question.
Name: Class: Date: Genetic Technology Multiple Choice Identify the choice that best completes the statement or answers the question. 1. An application of using DNA technology to help environmental scientists
More informationCloning Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems
Promega Notes Number 71, 1999, p. 10 Blunt-End Pfu DNA Polymerase- Generated PCR Fragments into pgem -T Vector Systems By Kimberly Knoche, Ph.D., and Dan Kephart, Ph.D. Promega Corporation Corresponding
More informationAviva Systems Biology
Aviva Custom Antibody Service and Price Mouse Monoclonal Antibody Service Package Number Description Package Contents Time Price Customer provides antigen protein $6,174 Monoclonal package1 (From protein
More informationHeterologous expression and purification of proteins in E. coli
Heterologous expression and purification of proteins in E. coli Rory Koenen Institut für molekulare Herz-Kreislaufforschung University Hospital of the RWTH Aachen rkoenen@ukaachen.de Tel. 35984 contents
More informationAviva Systems Biology
Aviva Custom Antibody Services and Prices Rabbit Polyclonal Antibody Service Package Number Description Package Contents Time Price Polyclonal package 1 (From protein to antiserum) Polyclonal package 2
More informationAntibody Production Price List
Antibody Production Price List Presenting Insight Biotechnology s price list for custom polyclonal and monoclonal antibody production services. We are happy to tailor individual packages towards the specific
More informationEuropean Medicines Agency
European Medicines Agency July 1996 CPMP/ICH/139/95 ICH Topic Q 5 B Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-dna Derived Protein
More informationMAB Solut. MABSolys Génopole Campus 1 5 rue Henri Desbruères 91030 Evry Cedex. www.mabsolut.com. is involved at each stage of your project
Mabsolus-2015-UK:Mise en page 1 03/07/15 14:13 Page1 Services provider Department of MABSolys from conception to validation MAB Solut is involved at each stage of your project Creation of antibodies Production
More informationBaculoDirect Baculovirus Expression System Free your hands with the BaculoDirect Baculovirus Expression System
BaculoDirect Baculovirus Expression System Free your hands with the BaculoDirect Baculovirus Expression System The BaculoDirect Baculovirus Expression System gives you: Unique speed and simplicity High-throughput
More informationClassic Immunoprecipitation
292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.
More informationLecture 1 MODULE 3 GENE EXPRESSION AND REGULATION OF GENE EXPRESSION. Professor Bharat Patel Office: Science 2, 2.36 Email: b.patel@griffith.edu.
Lecture 1 MODULE 3 GENE EXPRESSION AND REGULATION OF GENE EXPRESSION Professor Bharat Patel Office: Science 2, 2.36 Email: b.patel@griffith.edu.au What is Gene Expression & Gene Regulation? 1. Gene Expression
More informationName Class Date. Figure 13 1. 2. Which nucleotide in Figure 13 1 indicates the nucleic acid above is RNA? a. uracil c. cytosine b. guanine d.
13 Multiple Choice RNA and Protein Synthesis Chapter Test A Write the letter that best answers the question or completes the statement on the line provided. 1. Which of the following are found in both
More informationGenetic Engineering and Biotechnology
1 So, what is biotechnology?? The use of living organisms to carry out defined chemical processes for industrial or commercial application. The office of Technology Assessment of the U.S. Congress defines
More informationMolecular Cloning, Product Brochure
, Product Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0)26 326 44 50 T BE +32 (0)2 503 03 48 Begonialaan 3a F NL +31 (0)26 326 44 51 F BE
More informationComplex multicellular organisms are produced by cells that switch genes on and off during development.
Home Control of Gene Expression Gene Regulation Is Necessary? By switching genes off when they are not needed, cells can prevent resources from being wasted. There should be natural selection favoring
More informationAP Biology TEST #5 - Chapters 11-14, 16 - REVIEW SHEET
NAME: AP Biology TEST #5 - Chapters 11-14, 16 - REVIEW SHEET 1. Griffith's experiments showing the transformation of R strain pneumococcus bacteria to S strain pneumococcus bacteria in the presence of
More informationCLONING IN ESCHERICHIA COLI
CLONING IN ESCHERICHIA COLI Introduction: In this laboratory, you will carry out a simple cloning experiment in E. coli. Specifically, you will first create a recombinant DNA molecule by carrying out a
More informationRapid GST Inclusion Body Solubilization and Renaturation Kit
Product Manual Rapid GST Inclusion Body Solubilization and Renaturation Kit Catalog Number AKR-110 FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Bacteria are widely used for His
More informationDP419 RNAsimple Total RNA Kit. RNAprep pure Series. DP501 mircute mirna Isolation Kit. DP438 MagGene Viral DNA / RNA Kit. DP405 TRNzol Reagent
Overview of TIANGEN Products DP419 RNAsimple Total RNA Kit DP430 RNAprep pure Kit(For Cell/Bacteria) DP315/DP315-R TIANamp Virus DNA/RNA Kit DP431 RNAprep pure Kit (For Tissue) Silica-membrane Technology
More informationEffects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual
Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual I. Purpose...1 II. Introduction...1 III. Inhibition of Bacterial Growth Protocol...2 IV. Inhibition of in vitro
More information'LVFXVVLRQ $UUD\HGF'1$H[SUHVVLRQOLEUDULHV 5RERWWHFKQRORJ\DQGDUUD\HGOLEUDULHV
Discussion 74 'LVFXVVLRQ This study describes arrayed cdna libraries as a source of clonally expressed recombinant proteins which can be directly linked to clones characterised and identified by DNA hybridisation
More informationTrasposable elements: P elements
Trasposable elements: P elements In 1938 Marcus Rhodes provided the first genetic description of an unstable mutation, an allele of a gene required for the production of pigment in maize. This instability
More informationpmod2-puro A plasmid containing a synthetic Puromycin resistance gene Catalog # pmod2-puro For research use only Version # 11H29-MM
pmod2-puro A plasmid containing a synthetic Puromycin resistance gene Catalog # pmod2-puro For research use only Version # 11H29-MM PrOduct information content: - 20 mg of lyophilized pmod2-puro plasmid
More informationA and B are not absolutely linked. They could be far enough apart on the chromosome that they assort independently.
Name Section 7.014 Problem Set 5 Please print out this problem set and record your answers on the printed copy. Answers to this problem set are to be turned in to the box outside 68-120 by 5:00pm on Friday
More informationAP BIOLOGY 2007 SCORING GUIDELINES
AP BIOLOGY 2007 SCORING GUIDELINES Question 4 A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to completion with two restriction enzymes in three separate treatments: EcoRI, HaeIII,
More informationGenetics 301 Sample Final Examination Spring 2003
Genetics 301 Sample Final Examination Spring 2003 50 Multiple Choice Questions-(Choose the best answer) 1. A cross between two true breeding lines one with dark blue flowers and one with bright white flowers
More informationJust the Facts: A Basic Introduction to the Science Underlying NCBI Resources
1 of 8 11/7/2004 11:00 AM National Center for Biotechnology Information About NCBI NCBI at a Glance A Science Primer Human Genome Resources Model Organisms Guide Outreach and Education Databases and Tools
More informationHiPer RT-PCR Teaching Kit
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
More informationAbout Our Products. Blood Products. Purified Infectious/Inactivated Agents. Native & Recombinant Viral Proteins. DNA Controls and Primers for PCR
About Our Products Purified Infectious/Inactivated Agents ABI produces a variety of specialized reagents, allowing researchers to choose the best preparations for their studies. Available reagents include
More informationViruses. Viral components: Capsid. Chapter 10: Viruses. Viral components: Nucleic Acid. Viral components: Envelope
Viruses Chapter 10: Viruses Lecture Exam #3 Wednesday, November 22 nd (This lecture WILL be on Exam #3) Dr. Amy Rogers Office Hours: MW 9-10 AM Too small to see with a light microscope Visible with electron
More informationInnate Immunity. Insects rely solely on an innate immune system for defense against infection
Innate Immunity Insects rely solely on an innate immune system for defense against infection The importance of mammalian innate immunity has only recently become appreciated The innate immune response
More informationTranscription in prokaryotes. Elongation and termination
Transcription in prokaryotes Elongation and termination After initiation the σ factor leaves the scene. Core polymerase is conducting the elongation of the chain. The core polymerase contains main nucleotide
More informationRNA Viruses. A Practical Approac h. Alan J. Cann
RNA Viruses A Practical Approac h Alan J. Cann List of protocols page xiii Abbreviations xvii Investigation of RNA virus genome structure 1 A j. Easton, A.C. Marriott and C.R. Pringl e 1 Introduction-the
More informationSample Questions for Exam 3
Sample Questions for Exam 3 1. All of the following occur during prometaphase of mitosis in animal cells except a. the centrioles move toward opposite poles. b. the nucleolus can no longer be seen. c.
More informationThe world of non-coding RNA. Espen Enerly
The world of non-coding RNA Espen Enerly ncrna in general Different groups Small RNAs Outline mirnas and sirnas Speculations Common for all ncrna Per def.: never translated Not spurious transcripts Always/often
More informationBasic Concepts Recombinant DNA Use with Chapter 13, Section 13.2
Name Date lass Master 19 Basic oncepts Recombinant DN Use with hapter, Section.2 Formation of Recombinant DN ut leavage Splicing opyright lencoe/mcraw-hill, a division of he Mcraw-Hill ompanies, Inc. Bacterial
More informationFinal Review. Aptamers. Making Aptamers: SELEX 6/3/2011. sirna and mirna. Central Dogma. RNAi: A translation regulation mechanism.
Central Dogma Final Review Section Week 10 DNA RNA Protein DNA DNA replication DNA RNA transcription RNA Protein translation **RNA DNA reverse transcription http://bass.bio.uci.edu/~hudel/bs99a/lecture20/lecture1_1.html
More information3 months 1.5 months 1.5 months. 1 month
Rabbit monoclonal antibody (Mab) is secreted by the plasma B-cell of the rabbit. Traditional generation of rabbit Mab relies on a rabbit myeloma for B- cell fusion (
More informationHow many of you have checked out the web site on protein-dna interactions?
How many of you have checked out the web site on protein-dna interactions? Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. Find and be ready to discuss
More informationGenomic DNA Clean & Concentrator Catalog Nos. D4010 & D4011
Page 0 INSTRUCTION MANUAL Catalog Nos. D4010 & D4011 Highlights Quick (5 minute) spin column recovery of large-sized DNA (e.g., genomic, mitochondrial, plasmid (BAC/PAC), viral, phage, (wga)dna, etc.)
More informationRIBOPROTECT. RNase Inhibitor RT33-020, RT33-100
RIBOPROTECT RT33-020, RT33-100 RT33-020, RT33-100 RIBOPROTECT The RIBOPROTECT is a recombinant protein isolated and purified from Escherichia coli. It inhibits ribonuclease (RNase) activity of enzymes
More informationChoose your optimal tools for protein studies
Protein Purification Choose your optimal tools for protein studies Bacterial Baculoviral Cell free Mammalian Secreted Intracellular High yield Increased solubility Highest purity Highest yield His-tag
More informationGene Transcription in Prokaryotes
Gene Transcription in Prokaryotes Operons: in prokaryotes, genes that encode protein participating in a common pathway are organized together. This group of genes, arranged in tandem, is called an OPERON.
More informationGRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps)
1 GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) (FOR RESEARCH ONLY) Sample : Expected Yield : Endotoxin: Format : Operation Time : Elution Volume : 50-400 ml of cultured bacterial
More informationIMBB 2013. Genomic DNA purifica8on
IMBB 2013 Genomic DNA purifica8on Why purify DNA? The purpose of DNA purifica8on from the cell/8ssue is to ensure it performs well in subsequent downstream applica8ons, e.g. Polymerase Chain Reac8on (PCR),
More informationGene Regulation -- The Lac Operon
Gene Regulation -- The Lac Operon Specific proteins are present in different tissues and some appear only at certain times during development. All cells of a higher organism have the full set of genes:
More informationChapter 18 Regulation of Gene Expression
Chapter 18 Regulation of Gene Expression 18.1. Gene Regulation Is Necessary By switching genes off when they are not needed, cells can prevent resources from being wasted. There should be natural selection
More informationLecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology
Lecture 13: DNA Technology DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology DNA Sequencing determine order of nucleotides in a strand of DNA > bases = A,
More informationDNA (genetic information in genes) RNA (copies of genes) proteins (functional molecules) directionality along the backbone 5 (phosphate) to 3 (OH)
DNA, RNA, replication, translation, and transcription Overview Recall the central dogma of biology: DNA (genetic information in genes) RNA (copies of genes) proteins (functional molecules) DNA structure
More informationGreen Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein
Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein INTRODUCTION Green Fluorescent Protein (GFP) is a novel protein produced by the bioluminescent
More informationAdvanced BioDesign Outlines Solutions. Antibody Overview. by Advanced BioDesign. Project Start. Immunogenicity. Selecting Your Antigen
Advanced BioDesign Outlines Solutions by Advanced BioDesign Antibody Overview Launching an immunisation programme is an important experimental step that needs care. With Advanced BioDesign, you may develop
More informationGenetics Lecture Notes 7.03 2005. Lectures 1 2
Genetics Lecture Notes 7.03 2005 Lectures 1 2 Lecture 1 We will begin this course with the question: What is a gene? This question will take us four lectures to answer because there are actually several
More informationControl of Gene Expression
Home Gene Regulation Is Necessary? Control of Gene Expression By switching genes off when they are not needed, cells can prevent resources from being wasted. There should be natural selection favoring
More informationCHAPTER 6 GRIFFITH/HERSHEY/CHASE: DNA IS THE GENETIC MATERIAL IDENTIFICATION OF DNA DNA AND HEREDITY DNA CAN GENETICALLY TRANSFORM CELLS
CHAPTER 6 GRIFFITH/HERSHEY/CHASE: DNA IS THE GENETIC MATERIAL In 1928, Frederick Griffith was able to transform harmless bacteria into virulent pathogens with an extract that Oswald Avery proved, in 1944,
More informationQuestion 4 /29 points. Total /100 points
MIT Department of Biology 7.28, Spring 2005 - Molecular Biology 7.28 Spring 2005 Exam Three Question 1 Question 2 Question 3 /30 points /20 points /21 points Question 4 /29 points Total /100 points 1 Question
More informationCustom Antibodies Services. GeneCust Europe. GeneCust Europe
GeneCust Europe Laboratoire de Biotechnologie du Luxembourg S.A. 6 rue Dominique Lang L-3505 Dudelange Luxembourg Tél. : +352 27620411 Fax : +352 27620412 Email : info@genecust.com Web : www.genecust.com
More information