1 Gene Cloning 2004 Seungwook Kim Chem. & Bio. Eng. Reference T.A. Brown, Gene Cloning, Chapman and Hall S.B. Primrose, Molecular Biotechnology, Blackwell
2 Why Gene Cloning is Important? A century ago, Gregor Mendel : Basic assumption (each heritable property of an organism) is controlled by a factor (gene). In 1900, Mandel's law the birth of genetics. what these genes are and exactly how they work The Early Development of Genetics In 1903, Sutton, W Proposed that genes reside on chromosomes
3 In 1910, Morgan, TH Experimental backing on that --> > development of the techniques for gene mapping (To establish the structure or structural details or location) By 1922, a comprehensive analysis of the relative positions of over 2000 genes on the four chromosomes of the fruit fly. (Drosophilia melanogaster) In 1944, Avery, MacLeod and McCarty In 1952, Hershey and Chase Experimental results were shown that DNA is the genetic material. Conventional idea : genes were made of protein In , Delbruck, Chargaff, Crick and Monod The structure of DNA was elucidated. The genetic code was cracked. The process of transcription and translation were described.
4 The Advent of Gene Cloning In the late 1960's ; The experimental techniques were not sophisticated. In 1971 ~ 1973 ; A new experimental techniques were developed. Recombinant DNA technology or Genetic engineering based on the process of gene cloning This led to rapid and efficient DNA sequencing techniques that enabled the structures of individual genes to be determined. In the 1990s ; started with massive genome sequencing projects including the human project. This led to procedures for studying the regulation of individual genes.
5 Genome : A complete single set of the genetic material of a cell or of an organism. From the techniques, modern biotechnology is appeared for the production of proteins and other compounds needed in medicine and industrial processes. Introduction to Gene Cloning Let's suppose that we wish to construct a bacterium that produces human insulin. It might be thought that all that is required is to introduce the human insulin gene into its new host. Case A : The enzyme DNA polymerase, which makes copies of the DNA, does not initiate the process at random but at selected sites known as origins of replication. Replication: The process whereby a new daughter DNA molecule is synthesized from a parent DNA molecule. Case B : Recombinant DNA technology - Replication of the insulin gene in its new host by inserting the gene into a cloning vector. A cloning vector is simply a DNA molecule possessing an origin of replication and which can replicate in the host cell of choice.
6 Basic Steps of Gene Cloning
7 Basic Steps of Gene Cloning 1)A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule (vector) "Recombinant DNA molecule" or "Chimera" 2)The vector acts as a vehicle that transports the gene into a host cell (usually, bacterium) possibly other types of living cell. 3)Within the host cell the vector multiplies, producing numerous identical copies not only of itself but also of the gene that it carries. Basic Steps of Gene Cloning 4) When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place. 5) After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule. The gene is cloned.
8 Cloning vehicles & Clone Cloning vehicles DNA molecule must be capable of entering a host cell and replicating to produce multiple copies of itself. Clone A group of cells derived from a single parental cell by asexual reproduction. Plasmids Two naturally occurring types of DNA molecule A small circles of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome. Virus chromosomes The chromosomes of bacterophages (viruses that specifically infect bacteria) During infection, the bacteriophage DNA molecule is injected into the host cell where it undergoes replication.
9 Basic Skills for Handling DNA 1. Preparation of pure samples of DNA (cloning vehicle and the gene) 1. Cutting DNA molecules (cut a specific point) ; purified restriction endonucleases 2. Analysis of DNA fragment sizes 3. Joining DNA molecules together ; purified ligases 4. Introduction of DNA into host cells 5. Identification of cells that contain recombinant DNA molecules The Diversity of Cloning Vectors A wide variety of different cloning vectors are available. All are derived from naturally occuring plasmids or viruses. Those could be modified in various ways.
10 Why Gene Cloning is so Important? Gene isolation by cloning Cloning can provide a pure sample of an individual gene. The Problem of selection of just one gene. Only cells containing the desired recombinant DNA molecule can devide and the clone of interest is automatically selected.
11 Restriction Endonucleases. In order to insert foreign DNA into a plasmid, use is made of special enzymes known as restriction endonucleases. Restriction endonucleases These enzymes cut large DNA molecules into shorter fragments by cleavage at specific nucleotide sequences called regognition sites. These enzymes are highly specific deoxyribonucleases (DNases). The recognition sites for some common restriction endonucleases.
12 Examples of Restriction Enzymes Restriction Enzyme Action of EcoRI
13 Insertion of a DNA Sample into a plasmid Fragments of the foreign DNA are inserted into plasmid vectors cut open with the same enzyme or one which produces a match end.
14 Sticky end & Blunt end Sticky end : Some enzymes cut the two helices a few base pairs apart, generating two fragments with single-strand protrusions called sticky end. Blunt end (Flush end) : Some enzymes make a simple double-stranded cut in the middle of the recognition sequence. Three plasmid vectors used for gene cloning a. The structure of pbr322, a vector used with E. coli. b. The structure of phv33, a vector which can replicate in both E. coli and B. subtilis. c. The structure of YRp17, a vector which can replicate in both E. coli and yeast.
15 The Problem of Selection a. Even the simplest organisms, such as E. coli, contain several thousand genes, and a restriction digest of total cell DNA will produce not only the fragment carrying the desired gene, but also many other fragments carrying all the other genes. b. During the ligation reaction there is of course no selection for an individual fragment: numerous different recombinant DNA molecules are produced, all containing different pieces of DNA. c. A variety of recombinant clones are obtained after transformation and plating out. After the correct one must be identified. Two Basic Strategies for obtaining the Correct Clone Direct selection Clone identification
16 Direct Selection Plating-out method : only the correct recombinant can survive. The simplest example of direct selection occurs when the desired gene specifies resistance to an antibiotic (kanamycin). An experiment to clone the gene for kanamycin resistance from plasmid R 6-5 to pbr322 a. This plasmid carries genes for resistances to several antibiotics. The kanamycin resistance gene lies within one of the 13 EcoRI fragments. b. To clone this gene the EcoRI fragments of R6-5 would be inserted into the EcoRI site of a vector such as pbr322. c. In this case, kanamycin resistance gene can be used as the selectable marker. Transformants are plated on to kanamycin agar, on which the only cells able to survive and produce colonies are those recombinants that contain the cloned kanamycin resistance gene.
17 An experiment to clone the gene trpa from E. coli. This gene codes for the enzyme tryptophan synthase, which is involved in biosynthesis of the essential amino acid tryptophan. trpa- : a mutant strain of E. coli that has a nonfunctional trpa gene and is able to survive only if tryptophan is added to the th di a. E. coli mutant can be used to clone the correct version of the trpa gene. Digestion with a restriction endonuclease, followed by ligation into a vector, produces numerous recombinant DNA molecules, one of which may, with luck, carry an intact copy of the trpa gene(functional gene). b. The ligation mixture is used to transform the auxotrophic E. coli trpa- cells. c. The vast majority of the resulting transformants will be auxotrophic, but a few have the plasmid-borne copy of the correct trgene. These recombinants are non-auxotrophic; they no longer require tryptophan as the cloned gene is able to direct production of tryptophan synthase. d. Direct selection is performed by plating transformants onto minimal medium, which has no tryptophan. Auxotrophs cannot grow on minimal medium, so the only colonies to appear are recombinants that contain the cloned trpa gene.
18 Identification of the Clone From a Gene Library Analysis of the individual clones to identify the correct one. Problems of marker rescue; Many bacterial mutants are not auxotrophs. Foreign genes (from animals and plants) sometimes do not function in the bacterial cell. The Polymerase Chain Reaction (PCR) In 1985, Kary Mullis invented the polymerase chain reaction (PCR) What is PCR? PCR is carried out in a single test tube simply by mixing DNA with a set of reagents and placing the tube in a thermal cycler, a piece of equipment that enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner.
19 The basic steps in the PCR Basic Steps in PCR 1) The mixture is heated to 94 C, at which temperature the hydrogen bonds that hold together the two strands of the double-stranded DNA molecule are broken, causing the molecule to denature. 2) The mixture is cooled down to C. the two strands of each molecule could join back together at this temperature, but most do not because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific positions. 3) The temperature is raised to 74 C. This is the optimum working temperature for the Taq DNA polymerase that is present in the mixture. This enzyme attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules, during this step of the PCR. 4) The temperature is increased back to 94 C. The double-stranded DNA molecules, each of which consists of one strand of the original molecule and one new strand of DNA, denature into single strands. By repeating the cycle 25 times the doublestraned molecule that we began with is converted into over 50million new double-stranded molecules.
20 Gene Isolation by PCR The PCR can also be used to obtain a pure sample of a gene. If the primers anneal either side of the gene of interest, many copies of that gene will be synthesized. A PCR experiment can be completed in a few hours, whereas it takes weeks if not months to obtain a gene by cloning. Why then is Gene Cloning Still Used? 1) It is easy to synthesize a primer with a preddetermined sequence, but if the sequences of the annealing sites are unknown then the appropriate primers cannot be made. In this case, PCR cannot be used to isolate genes and that has to be done by cloning. 2) There is a limit to the length of DNA sequence that can be copied by PCR. Five kilobases(kb) can be copied fairly easily, and segments to 40kb can be dealt with using specialized techniques, but this is shorter than the lengths of many genes, especially those of humans and other vertebrates. Cloning must be used for long gene. Even if the sequence of a gene is not known, it may still be possible to determine the appropriate sequences for a pair of primers, based on what is known about the sequence of the equivalent gene in a different organism.
21 The Impact of Cloning on Research and Biotechnology Gene cloning, PCR, the recombinant techniques provide a way in which mammalian proteins can be produced in bacterial cells. Vehicles : Plasmids and Bacteriophages Numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells. A cloning vehicle needs to be relatively small, ideally less than 10kb in size; large molecules tend to break down during purification and also more difficult to manipulate. The most important type : plasmids and bacteriophage chromosomes.
22 Basic Features of Plasmids Plasmids : a circular molecules of DNA that lead to an independent existence in the bacterial cell. It carries antibiotic resistance genes. In the Lab., it was used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid.
23 All plasmids posses at least one DNA sequence that can act as an origin of replication ; multiply independently of the main bacterial chromosome (non-integrative plasmid) The smaller plasmids make use of the host cell's own DNA replicative enzymes in order to make copies of themselves. The larger plasmids carry genes that code for special enzymes that are specific for plasmid replication. A few types of plasmid are also able to replicate by inserting themselves the bacterial chromosome (Integrative plasmids : episomes) At some stage these plasmids exist as independent elements.
24 Size and Copy Number Plasmid range : 1 ~ 250kb Large plasmids may be adapted for cloning under some circumstances. Copy number : the number of molecules of an individual plasmid that are normally found in a single bacterial cell. Conjugation and Compatibility Conjugative plasmids The ability to promote sexual conjugation between bacterial cells. Conjugation and plasmid transfer are controlled by a set of transfer or tra genes. Non-conjugative plasmids Absence of tra genes Under some circumstances, it may be cotransferred along with a conjugative plasmid when both are present in the same cell.
25 Plasmid transfer by Conjugation between bacterial cells. Plasmid Classification It's based on the main characteristic coded by the plasmid genes. 1. Fertility or "F" plasmids carry only tra genes promote conjugal transfer of plasmids Ex) F plasmid of E. coli 2. Resistance or "R" plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents. Ex) RP4 in Pseudomonas 3. Col plasmids codes for colicins(proteins that kill other bacteria) Ex) ColE1 of E. coli 4. Degradative plasmids allows the host bacterium to metabolize unusual molecules such as toluene and salicylic acid Ex) TOL of Pseudomonas putida 5. Virulence plasmids confer pathogenicity on the host bacterium Ex) Ti plasmids of Agrobacterium tumefaciens
26 Eukaryotic Plasmid Saccharomyces cerevisiae 2 m circle However, It is suspected that many higher organisms simply do not harbour plasmids within their cells. Basic Features of Bacteriophages Viruses that specifically infect bacteria
27 The General Pattern of Infection The general pattern of infection is a three-step process. 1. The phage particle attaches to the outside of the bacterium and injects its DNA chromosome in to the cell. 2. The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes on the phage chromosome. 3. Other phage genes direct synthesis of the protein components of the capsid, and new phage particles are assembled and released from the bacterium. Lytic cycle The entire infection cycle takes less than 20 min. DNA replication is immediately followed by synthesis of capsid proteins, the phage DNA molecule is never maintained in a stable condition in the host cell.
28 Lysogenic Phages is characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions. Prophage The integrated form of the phage DNA (the phage DNA is inserted into the bacterial genome, similar to episomal insertion) Lysogen A baterium (carries a prophage) usually physiologically indistinguishable from an uninfected cell. Prophage is eventually released from the host genome and the phage reverts to the lytic mode and lyses the cell. The lysogenic infection cycle of bacteriophage
29 The infection cycle of bacteriophage M13 The M13 DNA is not integrated into the bacterial genome. Cell lysis never occurs.
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
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
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
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
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
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
DNA Scissors: Introduction to Restriction Enzymes Objectives At the end of this activity, students should be able to 1. Describe a typical restriction site as a 4- or 6-base- pair palindrome; 2. Describe
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
The E. coli Insulin Factory BACKGROUND Bacteria have not only their normal DNA, they also have pieces of circular DNA called plasmids. Plasmids are a wonderfully ally for biologists who desire to get bacteria
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
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
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.
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
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
Chem 121 Chapter 22. Nucleic Acids 1. Any given nucleotide in a nucleic acid contains A) two bases and a sugar. B) one sugar, two bases and one phosphate. C) two sugars and one phosphate. D) one sugar,
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
Chapter 6 DNA Replication Each strand of the DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. Each strand can therefore
Viruses VIRUSES Viruses are small packages of genes Consist of protein coat around nucleic acids ( or RNA) Viruses measured in nanometers (nm). Require electron microscopy. Obligate intracellular parasites
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,
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,
How was DNA shown to be the genetic material? We need to discuss this in an historical context. During the 19th century most scientists thought that a bit of the essence of each and every body part was
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.
Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection
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
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
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
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,
Fact Sheet Describing Recombinant DNA and Elements Utilizing Recombinant DNA Such as Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology Compiled and/or written
DNA Technology Mapping a plasmid A first step in working with DNA is mapping the DNA molecule. One way to do this is to use restriction enzymes (restriction endonucleases) that are naturally found in bacteria
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
Chapter 11: Molecular Structure of DNA and RNA Student Learning Objectives Upon completion of this chapter you should be able to: 1. Understand the major experiments that led to the discovery of DNA as
Genetics Module B, Anchor 3 Key Concepts: - An individual s characteristics are determines by factors that are passed from one parental generation to the next. - During gamete formation, the alleles for
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,
Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins
Bio 102 Practice Problems Recombinant DNA and Biotechnology Multiple choice: Unless otherwise directed, circle the one best answer: 1. Which of the following DNA sequences could be the recognition site
IIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR) Background Infectious diseases caused by pathogenic organisms such as bacteria, viruses, protozoa,
Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction Objectives Understand the process of DNA isolation Perform DNA isolation using cheek cells Use thermal cycler and Taq polymerase to perform DNA
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.
Nucleotides and Nucleic Acids Brief History 1 1869 - Miescher Isolated nuclein from soiled bandages 1902 - Garrod Studied rare genetic disorder: Alkaptonuria; concluded that specific gene is associated
A Genomic Timeline Tim Shank 2003 1800s 1865 Gregor Mendel reports the results of his pea plant expts, from which he discerned several fundamental laws of heredity. His results appeared in an obscure journal
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
Amazing DNA facts These facts can form the basis of a quiz (for example, how many base pairs are there in the human genome?). Students should be familiar with most of this material, so the quiz could be
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
Transmission of genetic variation: conjugation Transmission of genetic variation: conjugation Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium
OpenStax-CNX module: m44488 1 DNA Replication in Prokaryotes OpenStax College This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 3.0 By the end of this section,
Protocol for Cloning GFP into Mammalian cells Studiepraktik 2013 Molecular Biology and Molecular Medicine Aarhus University Produced by the instructors: Tobias Holm Bønnelykke, Rikke Mouridsen, Steffan
Bio 102 Practice Problems Chromosomes and DNA Replication Multiple choice: Unless otherwise directed, circle the one best answer: 1. Which one of the following enzymes is NT a key player in the process
Chapter IV Molecular Computation These lecture notes are exclusively for the use of students in Prof. MacLennan s Unconventional Computation course. c 2013, B. J. MacLennan, EECS, University of Tennessee,
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
1. True or False? A typical chromosome can contain several hundred to several thousand genes, arranged in linear order along the DNA molecule present in the chromosome. True 2. True or False? The sequence
Gene Mapping Techniques OBJECTIVES By the end of this session the student should be able to: Define genetic linkage and recombinant frequency State how genetic distance may be estimated State how restriction
Cell Structure and Organization 1. All living things must possess certain characteristics. They are all composed of one or more cells. They can grow, reproduce, and pass their genes on to their offspring.
AS Unit 1: Basic Biochemistry and Cell Organisation Name: Date: Topic 1.5 Nucleic Acids and their functions Page 3 l. DNA Replication 1. Go through PowerPoint 2. Read notes p2 and then watch the animation
How is genome sequencing done? Using 454 Sequencing on the Genome Sequencer FLX System, DNA from a genome is converted into sequence data through four primary steps: Step One DNA sample preparation; Step
DNA Ligase enzyme catalysing formation of phosphodiesteric bound between group 3 -OH of one end of DNA molecule and group 5 -phosphate of the second end of DNA DNA ligase ATP (or NAD+) Ligase cofactors
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
BIOTECHNOLOGY I DNA RESTRICTION FOR CLONING LAB 7 DNA RESTRICTION for CLONING STUDENT GUIDE GOALS The goals of this lab are to provide the biotech student with experience in DNA digestion with restriction
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
BacReady TM Multiplex PCR System Technical Manual No. 0191 Version 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI Detailed Experimental
Investigating a Eukaryotic Genome: Cloning and Sequencing a Fragment of Yeast DNA Credits: This lab was created by Sarah C.R. Elgin and developed and written by Kathleen Weston-Hafer. Specific protocols
DNA, RNA, Protein Synthesis Keystone 1. During the process shown above, the two strands of one DNA molecule are unwound. Then, DNA polymerases add complementary nucleotides to each strand which results
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
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
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.
EDVTEK P.. Box 1232 West Bethesda, MD 20827-1232 The Biotechnology 106 EDV-Kit # Principles of DNA Sequencing Experiment bjective: The objective of this experiment is to develop an understanding of DNA
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
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),
An Overview of DNA Sequencing Prokaryotic DNA Plasmid http://en.wikipedia.org/wiki/image:prokaryote_cell_diagram.svg Eukaryotic DNA http://en.wikipedia.org/wiki/image:plant_cell_structure_svg.svg DNA Structure
Forensic DNA Testing Terminology ABI 310 Genetic Analyzer a capillary electrophoresis instrument used by forensic DNA laboratories to separate short tandem repeat (STR) loci on the basis of their size.
Transformation of the bacterium E. coli using a gene for Green Fluorescent Protein Background In molecular biology, transformation refers to a form of genetic exchange in which the genetic material carried
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
20.109 MOD1 DNA ENGINEERING Fall 2010 Exploiting science for engineering: BRCA2 targeted therapies Orsi Kiraly Engelward lab Homologous recombination is important No HR chromosomal aberrations cell death
Cat. # R010A For Research Use PrimeSTAR HS DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 Features...3 V. General Composition of PCR Reaction
Section 16.1 Producing DNA fragments Recombinant DNA combined DNA of two different organisms The process of using DNA technology to make certain proteins is as follows: 1.) Isolation of the DNA fragments
Section 17.1: The Linnaean System of Classification Unit 9 Study Guide KEY CONCEPT Organisms can be classified based on physical similarities. VOCABULARY taxonomy taxon binomial nomenclature genus MAIN
Description: Molecular Biology s and DNA Sequencing DNA Sequencing s Single Pass Sequencing Sequence data only, for plasmids or PCR products Plasmid DNA or PCR products Plasmid DNA: 20 100 ng/μl PCR Product:
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
Chapter 4 The role of mutation in evolution Objective Darwin pointed out the importance of variation in evolution. Without variation, there would be nothing for natural selection to act upon. Any change
Bio 102 Practice Problems Genetic Code and Mutation Multiple choice: Unless otherwise directed, circle the one best answer: 1. Beadle and Tatum mutagenized Neurospora to find strains that required arginine
LAB 16 Rapid Colony Transformation of E. coli with Plasmid DNA Objective: In this laboratory investigation, plasmids containing fragments of foreign DNA will be used to transform Escherichia coli cells,
DNA Replication & Protein Synthesis This isn t a baaaaaaaddd chapter!!! The Discovery of DNA s Structure Watson and Crick s discovery of DNA s structure was based on almost fifty years of research by other
GENETIC TRANSFORMATION OF BACTERIA WITH THE GENE FOR GREEN FLUORESCENT PROTEIN (GFP) LAB BAC3 Adapted from "Biotechnology Explorer pglo Bacterial Transformation Kit Instruction Manual". (Catalog No. 166-0003-EDU)