DOCSLIB.ORG

Genomics, Proteomics, and Genetic Engineering

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genomics, Proteomics, and Genetic Engineering Rice (Oryza sativa) is among the world’s most important food crops. With 12 pairs of chromosomes and about 40,000 genes in 400 Mb of DNA, its genome is one of the smallest of the major crop grasses. © Revensis/Dreamstime.com. Genomics, Proteomics, 10 and Genetic Engineering »» CHAPTER ORGANIZATION 10.1 Cloning a DNA molecule takes place in several 10.2 A genomic sequence is like a book without an index, steps. 332 and identifying genes and their functions is a major Restriction enzymes cleave DNA into fragments challenge. 343 with defined ends. 332 The protein-coding potential of an organism is Restriction fragments are joined end to end to contained in its genome sequence. 343 produce recombinant DNA. 334 A genome sequence without annotation is A vector is a carrier for recombinant DNA. 335 meaningless. 343 Specialized vectors can carry very large DNA Comparison among genomes is an aid to fragments. 336 annotation. 344 Vector and target DNA fragments are joined with 10.3 Genomics and proteomics reveal genome-wide DNA ligase. 337 A recombinant cDNA contains the coding sequence patterns of gene expression and networks of protein of a eukaryotic gene. 337 interactions. 347 Loss of b-galactosidase activity is often used to DNA microarrays are used to estimate the relative detect recombinant vectors. 339 level of gene expression of each gene in the Recombinant clones are often identified by genome. 347 hybridization with labeled probe. 342 Microarrays reveal groups of genes that are coordinately expressed during development. 348 Yeast two-hybrid analysis reveals networks of protein interactions. 350 331 773649_CH10_331_364.indd3649_CH10_331_364.indd 333131 99/17/09/17/09 22:22:22 PPMM 10.4 Reverse genetics creates an organism with a The production of useful proteins is a primary designed mutation. 352 impetus for recombinant DNA. 360 Animal viruses may prove useful vectors for gene Recombinant DNA can be introduced into the therapy. 360 germ line of animals. 353 Recombinant DNA can also be introduced into the human connection Pinch of This plant genomes. 356 and a Smidgen of That 355 Transformation rescue is used to determine experimentally the physical limits of a gene. 357 »» Chapter Summary 361 10.5 Genetic engineering is applied in medicine, industry, »» Issues & Ideas 361 agriculture, and research. 358 »» Solutions: Step by Step 362 Animal growth rate can be genetically »» Concepts in Action: Problems for Solution 362 engineered. 358 GENETICS on the web Crop plants with improved nutritional qualities can be created. 358 into a cell or organism to change its genotype in a ere is an incomplete list of mammalian directed, predetermined way. Such genetically engi- genomes that have been sequenced: human, neered organisms are called transgenic organisms. chimpanzee, monkey, lemur, mouse, dog, H Transgenics are often created for experimental stud- cat, bat, squirrel, rabbit, guinea pig, armadillo, ies, but an important application is the development hedgehog, shrew, opposum, horse, elephant, pango- of improved varieties of domesticated animals and lin, sloth, llama, and dolphin. Also sequenced are the crop plants, in which case a transgenic organism genomes of many species of fruit flies, worms, and is often called a genetically modified organism (GMO). fungi, hundreds of bacteria, mitochondria, and chlo- Specific examples of genetically modified organisms roplasts, and thousands of viruses. Together these are considered later in this chapter. genomes represent a colossal amount of sequence data available for analysis and comparison. In addi- tion to the genome sequences, methods are also 10.1 Cloning a DNA molecule takes available for identifying which genes in the genome place in several steps. are transcribed in particular tissue types, at specific times in development, or at different stages of the In genetic engineering, the immediate goal of an cell cycle. These are the raw data of genomics, experiment is usually to insert a particular fragment which deals with the DNA sequence, organization, of chromosomal DNA into a plasmid or a viral DNA function, and evolution of genomes. The counterpart molecule. This is accomplished by techniques for at the level of proteins is proteomics, which aims to breaking DNA molecules at specific sites and for iso- identify all the proteins in a cell or organism (includ- lating particular DNA fragments. ing any post-translationally modified forms), as well as their cellular localization, functions, and interac- Restriction enzymes cleave DNA into tions. Proteomics makes use of methods discussed fragments with defined ends. later in this chapter that identify which proteins in the cell undergo physical contact, thereby revealing DNA fragments are usually obtained by the treatment networks of interacting proteins. of DNA samples with restriction enzymes. Restriction Genomics was made possible by the invention of enzymes are nucleases that cleave DNA wherever it techniques originally devised for the manipulation contains a particular short sequence of nucleotides of genes and the creation of genetically engineered that matches the restriction site of the enzyme (see organisms with novel genotypes and phenotypes. Section 6.6). Most restriction sites consist of four or We refer to this approach as recombinant DNA, six nucleotides, within which the restriction enzyme but it also goes by the names gene cloning or genetic makes two single-strand breaks, one in each strand, engineering. The basic technique is quite simple: DNA generating 3Ј-OH and 5Ј-P groups at each position. is isolated and cut into fragments by one or more About a thousand restriction enzymes, nearly all restriction enzymes; then the fragments are joined with different restriction site specificities, have been together in a new combination and introduced back isolated from microorganisms. 332 CHAPTER 10 Genomics, Proteomics, and Genetic Engineering 773649_CH10_331_364.indd3649_CH10_331_364.indd 333232 99/17/09/17/09 22:22:22 PPMM Restriction sites for EcoRI GAATTC GAATTC GAATTC CTTAAG CTTAAG CTTAAG G AATTC G CTTAA G CTTAA ATTCA G AATTC G C TTAA G Any two Circularization “sticky” ends produced by Circularization the same A T A T A C restriction G T T A A T T G enzyme can G A C C T A come together C T A G and form base pairs. Circularization of DNA fragments produced by a restriction enzyme. The red arrowheads indicate the EcoRI cleavage sites. Most restriction sites are symmetrical in the sense • The breaks need not be directly opposite one that the sequence is identical in both strands of the another in the two DNA strands. DNA duplex. For example, the restriction enzyme • Enzymes that cleave the DNA strands asymmet- EcoRI, isolated from E. coli, has the restriction site rically generate DNA fragments with comple- 5Ј-GAATTC-3Ј; the sequence of the other strand is mentary ends. 3Ј-CTTAAG-5Ј, which is identical but written with the 3Ј end at the left. EcoRI cuts each strand between These properties are illustrated for EcoRI in . the G and the A. The term palindrome is used to Most restriction enzymes are like EcoRI in that denote this type of symmetrical sequence. they make staggered cuts in the DNA strands, pro- Soon after restriction enzymes were discovered, ducing single-stranded ends called sticky ends observations with the electron microscope indicated that can adhere to each other because they contain that the fragments produced by many restriction complementary nucleotide sequences. Some restric- enzymes could spontaneously form circles. The circles tion enzymes (such as EcoRI) leave a single-stranded Ј could be made linear again by heating. On the other overhang at the 5 end ( , part A); oth- Ј hand, if the circles that formed spontaneously were ers leave a 3 overhang. A number of restriction treated with DNA ligase, which joins 3Ј-OH and 5Ј-P enzymes cleave both DNA strands at the center of groups, then they could no longer be made linear with symmetry, forming blunt ends. Part B of Figure heat because the ends were covalently linked by the 10.2 shows the blunt ends produced by the enzyme DNA ligase. This observation was the first evidence for BalI. Blunt ends also can be ligated by DNA ligase. three important features of restriction enzymes: However, whereas ligation of sticky ends re-creates the original restriction site, any blunt end can join • Restriction enzymes cleave DNA molecules in with any other blunt end and not necessarily create palindromic sequences. a restriction site. 10.1 Cloning a DNA Molecule Takes Place in Several Steps 333 773649_CH10_331_364.indd3649_CH10_331_364.indd 333333 99/17/09/17/09 22:22:22 PPMM EcoRI restriction site Bal I restriction site (A) Cuts asymmetrically (B) Cuts on line of symmetry GAATTC TGGCCA CTTAAG ACCGGT When the cleavage is When the cleavage asymmetrical, the Separation of Separation of is symmetrical, the over-hanging ends are fragments fragments resulting ends are “sticky” (complementary). blunt. G ATTCA T G G C C A CTTAA G ACC GGT Sticky-end fragments Blunt-end fragments Two types of cuts made by restriction enzymes. The red arrowheads indicate the cleavage sites. (A) Cuts made in each strand at an equal distance from the center of symmetry of the restriction site. (B) Cuts made in each strand at the center of symmetry of the restriction site. Most restriction enzymes recognize their restric- tion sequence without regard to the source of the DNA. Thus, This principle will be seen to be one of the founda- tions of recombinant DNA technology. Because most restriction enzymes recognize a unique sequence, the number of cuts made in the DNA of an organism by a particular enzyme is limited. For example, an E. coli DNA molecule contains 4.6 106 base pairs, and any enzyme that cleaves a six-base restriction site will cut the molecule into about a thou- sand fragments.
Load more

Recommended publications
  • Restriction Endonucleases
    Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic) 9 Restriction Endonucleases Derek Robinson, Paul R. Walsh, and Joseph A. Bonventre Background Information . 226 Which Restriction Enzymes Are Commercially Available? . 226 Why Are Some Enzymes More Expensive Than Others? . 227 What Can You Do to Reduce the Cost of Working with Restriction Enzymes? . 228 If You Could Select among Several Restriction Enzymes for Your Application, What Criteria Should You Consider to Make the Most Appropriate Choice? . 229 What Are the General Properties of Restriction Endonucleases? . 232 What Insight Is Provided by a Restriction Enzyme’s Quality Control Data? . 233 How Stable Are Restriction Enzymes? . 236 How Stable Are Diluted Restriction Enzymes? . 236 Simple Digests . 236 How Should You Set up a Simple Restriction Digest? . 236 Is It Wise to Modify the Suggested Reaction Conditions? . 237 Complex Restriction Digestions . 239 How Can a Substrate Affect the Restriction Digest? . 239 Should You Alter the Reaction Volume and DNA Concentration? . 241 Double Digests: Simultaneous or Sequential? . 242 225 Genomic Digests . 244 When Preparing Genomic DNA for Southern Blotting, How Can You Determine If Complete Digestion Has Been Obtained? . 244 What Are Your Options If You Must Create Additional Rare or Unique Restriction Sites? . 247 Troubleshooting . 255 What Can Cause a Simple Restriction Digest to Fail? . 255 The Volume of Enzyme in the Vial Appears Very Low. Did Leakage Occur during Shipment? . 259 The Enzyme Shipment Sat on the Shipping Dock for Two Days.
    [Show full text]
  • Experiment 2 Plasmid DNA Isolation, Restriction Digestion and Gel Electrophoresis
    Experiment 2 Plasmid DNA Isolation, Restriction Digestion and Gel Electrophoresis Plasmid DNA isolation introduction: diatomaceous earth. After RNaseA treatment, The application of molecular biology techniques the DNA containing supernatant is bound to the to the analysis of complex genomes depends on diatomaceous earth in a chaotropic buffer, the ability to prepare pure plasmid DNA. Most often guanadine chloride or urea. The plasmid DNA isolation techniques come in two chaotropic buffer will force the silica flavors, simple - low quality DNA preparations (diatomaceous earth) to interact and more complex, time-consuming high quality DNA preparations. For many DNA manipulations such as restriction enzyme analysis, subcloning and agarose gel electrophoresis, the simple methods are sufficient. The high quality preparations are required for most DNA sequencing, PCR manipulations, transformation hydrophobically with the DNA. Purification using and other techniques. silica-technology is based on a simple bind- wash-elute procedure. Nucleic acids are The alkaline lysis preparation is the most adsorbed to the silica-gel membrane in the commonly used method for isolating small presence of high concentrations of amounts of plasmid DNA, often called minipreps. chaotropic salts, which remove water from This method uses SDS as a weak detergent to hydrated molecules in solution. Polysaccharides denature the cells in the presence of NaOH, and proteins do not adsorb and are removed. which acts to hydrolyze the cell wall and other After a wash step, pure nucleic acids are eluted cellular molecules. The high pH is neutralized by under low-salt conditions in small volumes, ready the addition of potassium acetate. The for immediate use without further concentration.
    [Show full text]
  • Gibson Assembly Cloning Guide, Second Edition
    Gibson Assembly® CLONING GUIDE 2ND EDITION RESTRICTION DIGESTFREE, SEAMLESS CLONING Applications, tools, and protocols for the Gibson Assembly® method: • Single Insert • Multiple Inserts • Site-Directed Mutagenesis #DNAMYWAY sgidna.com/gibson-assembly Foreword Contents Foreword The Gibson Assembly method has been an integral part of our work at Synthetic Genomics, Inc. and the J. Craig Venter Institute (JCVI) for nearly a decade, enabling us to synthesize a complete bacterial genome in 2008, create the first synthetic cell in 2010, and generate a minimal bacterial genome in 2016. These studies form the framework for basic research in understanding the fundamental principles of cellular function and the precise function of essential genes. Additionally, synthetic cells can potentially be harnessed for commercial applications which could offer great benefits to society through the renewable and sustainable production of therapeutics, biofuels, and biobased textiles. In 2004, JCVI had embarked on a quest to synthesize genome-sized DNA and needed to develop the tools to make this possible. When I first learned that JCVI was attempting to create a synthetic cell, I truly understood the significance and reached out to Hamilton (Ham) Smith, who leads the Synthetic Biology Group at JCVI. I joined Ham’s team as a postdoctoral fellow and the development of Gibson Assembly began as I started investigating methods that would allow overlapping DNA fragments to be assembled toward the goal of generating genome- sized DNA. Over time, we had multiple methods in place for assembling DNA molecules by in vitro recombination, including the method that would later come to be known as Gibson Assembly.
    [Show full text]
  • Cloning of Gene Coding Glyceraldehyde-3-Phosphate Dehydrogenase Using Puc18 Vector
    Available online a t www.pelagiaresearchlibrary.com Pelagia Research Library European Journal of Experimental Biology, 2015, 5(3):52-57 ISSN: 2248 –9215 CODEN (USA): EJEBAU Cloning of gene coding glyceraldehyde-3-phosphate dehydrogenase using puc18 vector Manoj Kumar Dooda, Akhilesh Kushwaha *, Aquib Hasan and Manish Kushwaha Institute of Transgene Life Sciences, Lucknow (U.P), India _____________________________________________________________________________________________ ABSTRACT The term recombinant DNA technology, DNA cloning, molecular cloning, or gene cloning all refers to the same process. Gene cloning is a set of experimental methods in molecular biology and useful in many areas of research and for biomedical applications. It is the production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques. cDNA is synthesized by using template RNA isolated from blood sample (human). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Amplify the gene (GAPDH) using primer forward and reverse with the sequence of 5’-TGATGACATCAAGAAGGTGGTGAA-3’ and 5’-TCCTTGGAGGCCATGTGGGCCAT- 3’.pUC18 high copy cloning vector for replication in E. coli, suitable for “blue-white screening” technique and cleaved with the help of SmaI restriction enzyme. Modern cloning vectors include selectable markers (most frequently antibiotic resistant marker) that allow only cells in which the vector but necessarily the insert has been transfected to grow. Additionally the cloning vectors may contain color selection markers which provide blue/white screening (i.e. alpha complementation) on X- Gal and IPTG containing medium. Keywords: RNA isolation; TRIzol method; Gene cloning; Blue/white screening; Agarose gel electrophoresis.
    [Show full text]
  • Digestion of DNA with Restriction Endonucleases 3.1.2
    RESTRICTION ENDONUCLEASES SECTION I Digestion of DNA with Restriction UNIT 3.1 Endonucleases Restriction endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease (see Basic Protocol). A number of common applications of this technique are also described. These include digesting a given DNA sample with more than one endonuclease (see Alternate Protocol 1), digesting multiple DNA samples with the same endonuclease (see Alternate Protocol 2), and partially digesting DNA such that cleavage only occurs at a subset of the restriction sites (see Alternate Protocol 3). A protocol for methylating specific DNA sequences and protecting them from restriction endonuclease cleavage is also presented (see Support Protocol). A collection of tables describing restriction endonucleases and their properties (including information about recognition sequences, types of termini produced, buffer conditions, and conditions for thermal inactivation) is given at the end of this unit (see Table 3.1.1, Table 3.1.2, Table 3.1.3, and Table 3.1.4). DIGESTING A SINGLE DNA SAMPLE WITH A SINGLE RESTRICTION BASIC ENDONUCLEASE PROTOCOL Restriction endonuclease cleavage is accomplished simply by incubating the enzyme(s) with the DNA in appropriate reaction conditions. The amounts of enzyme and DNA, the buffer and ionic concentrations, and the temperature and duration of the reaction will vary depending upon the specific application.
    [Show full text]
  • GFP Plasmid) 35X
    Název: Biotechnology and Fluorescent protein Školitel: Ana Maria Jimenez Jimenez, Iva Blažková Datum: 19.7.2013 Reg.č.projektu: CZ.1.07/2.3.00/20.0148 Název projektu: Mezinárodní spolupráce v oblasti "in vivo" zobrazovacích technik INTRODUCTION GFP - Green fluorescent protein A protein composed of 238 amino acid residues (26.9 kDa) exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range GFP was isolated from the jellyfish Aequorea victoria. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression The GFP gene has been introduced and expressed in many bacteria, yeast and other fungi, fish, plant and mammalian cells. Green fluorescent protein (GFP) Nobel prize in Chemistry (2008): for the discovery and development of the green fluorescent protein, GFP Osamu Shimomura Martin Chalfie Roger Y. Tsien Now GFP is found in laboratories all over the world where it is used in every conceivable plant and animal GFP Types Fluorescent proteins enable the creation of highly specific biosensors to monitor a wide range of intracellular phenomena. Mutagenesis of A. victoria GFP has resulted in fluorescent proteins that range in color from blue to yellow Transluminator Fluoresence spectrophotometry Fluorescence In-vivo Xtreme Detection Fluorescence microscopy In-vivo Xtreme cm Polyacrylamide gel In-vivo Xtreme 1/16 1/8 1/4 1/2 1 10000 y = 9003x + 732,46 9000 R² = 0,9984 Max-Backgroumd 8000 7000 Rostoucí koncentrace Mean-Background 6000 5000 4000 3000 y = 5748,2x + 490,64 R² = 0,9995 2000 Fluorescence intensity [a.u.] 1000 0 0 0,2 0,4 0,6 0,8 1 1,2 concentration [µg/ml] In-vivo Xtreme GFP encapsulated GFP in liposome GFP water in liposome Max.int: 8800 a.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • Restriction Digest
    PROTOCOL 3: RESTRICTION DIGEST TEACHER VERSION THE GENOME TEACHING GENERATION PROTOCOL 3: RESTRICTION DIGEST PROTOCOL 3: RESTRICTION DIGEST TEACHER VERSION PRE-REQUISITES & GOALS STUDENT PRE-REQUISITES Prior to implementing this lab, students should understand: • The central dogma of how DNA bases code for mRNA and then for proteins • How DNA samples were collected and prepared for PCR • The steps that occur during the process of polymerase change reaction (PCR) • What restriction enzymes are and how they work • How the sequence variants in OXTR and CYP2C19 are affected by restriction enzyme digestion • The purpose of PROTOCOL 3 is to determine genotype STUDENT LEARNING GOALS 1. Perform restriction digestion of PCR products of CYP2C19 and/or OXTR. 2. Describe the possible genotypes for individuals with the CYP2C19 and/or OXTR genes. 3. Predict what each genotype will look like after gel electrophoresis and why. 2 TEACHING THE GENOME GENERATION | THE JACKSON LABORATORY PROTOCOL 3: RESTRICTION DIGEST TEACHER VERSION CURRICULUM INTEGRATION Use the planning notes space provided to reflect on how this protocol will be integrated into your classroom. You’ll find every course is different, and you may need to make changes in your preparation or set-up depending on which course you are teaching. Course name: 1. What prior knowledge do the students need? 2. How much time will this lesson take? 3. What materials do I need to prepare in advance? 4. Will the students work independently, in pairs, or in small groups? 5. What might be challenge points
    [Show full text]
  • SNP2RFLP: a Computational Tool to Facilitate Genetic Mapping Using Benchtop Analysis of Snps
    Mamm Genome (2008) 19:687–690 DOI 10.1007/s00335-008-9149-2 SNP2RFLP: a computational tool to facilitate genetic mapping using benchtop analysis of SNPs Wesley A. Beckstead Æ Bryan C. Bjork Æ Rolf W. Stottmann Æ Shamil Sunyaev Æ David R. Beier Received: 9 September 2008 / Accepted: 24 September 2008 / Published online: 29 October 2008 Ó Springer Science+Business Media, LLC 2008 Abstract Genome-wide analysis of single nucleotide Introduction polymorphism (SNP) markers is an extremely efficient means for genetic mapping of mutations or traits in mice. The positional cloning and characterization of mutations in However, this approach often defines a relatively large the mouse is a powerful means for functional annotation of recombinant interval. To facilitate the refinement of this the mammalian genome. Many mouse gene mutations interval, we developed the program SNP2RFLP. This cause phenotypes that serve as models of human genetic program can be used to identify region-specific SNPs in disorders. Mapping and positional cloning of these poten- which the polymorphic nucleotide creates a restriction tially accelerate our understanding of the mouse gene, its fragment length polymorphism (RFLP) that can be readily human ortholog, and the underlying etiology of the disor- assayed at the benchtop using restriction enzyme digestion der. The utilization of single nucleotide polymorphism of SNP-containing PCR products. The program permits (SNP) markers has markedly facilitated genetic mapping user-defined queries that maximize the informative mark- because they are abundant throughout the genome and can ers for a particular application. This facilitates fine- be analyzed in a high-throughput manner using automated mapping in a region containing a mutation of interest, technology (Wang et al.
    [Show full text]
  • Easy Subcloning Prot
    Easy Subcloning by Michael Koelle Subcloning should be easy and fast, and work every time. The following protocols minimize the number of manipulations required to prepare DNA fragments for ligations, thereby both saving time and increasing reliability. Preparation of DNA fragments for ligation. 1. Restriction digests: Always cut a lot of your starting plasmids in a small volume; this will help in the gel purification of your restriction fragments by giving you a high concentration of DNA compared to agarose in your gel slice.. About 1 µg in a 20 µl reaction is good. For double digests: you almost never have to digest with one enzyme, adjust the buffer, and digest with the second. Look in the Biolabs catalogue to find a compromise buffer, and save yourself some time. Don't use just 1 unit of enzyme and wait an hour: your time is worth too much, and the enzymes are cheap and pure. Use about a 10-fold enzyme excess and digest about 30 min. 2. Playing with the ends. Blunting 5' overhangs: Add 1 µl 2 mM all four dNTPs to your 20 µl restriction digest. Add 0.5-1 µl Klenow (2-5 units), and incubate at room temp for 30 min. Blunting 3' overhangs: Add 1 µl 2 mM all four dNTPs to your 20 µl restriction digest. Add 0.5-1 µl T4 DNA polymerase, and incubate at 37° for 5 min. T4 polymerase has a more active 3' to 5' exo activity than Klenow, and so is preferred for this reaction, but Klenow will work.
    [Show full text]
  • Isolating Microsatelline DNA Loci*
    Isolating Microsatelline DNA Loci* Travis C. Glenn1,2 and Nancy A. Schable1,2 1Savannah River Ecology Laboratory, University of Georgia, Drawer E, Aiken, SC 29802, USA 2Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA Contact: Travis Glenn: Phone 803-725-5746; Fax 803-725-3309; E-mail: [email protected] Abstract A series of techniques are presented to construct genomic DNA libraries highly enriched for microsatellite DNA loci. The individual techniques used here derive from several published protocols, but have been optimized and tested in our research labs as well as classroom settings at the University of South Carolina and University of Georgia, with students achieving nearly 100% success. Reducing the number of manipulations involved has been a key to success, decreasing both the failure rate and the time necessary to isolate loci of interest. These protocols have been successfully used in our lab to isolate microsatellite DNA loci from more than 125 species representing all eukaryotic kingdoms. Using these protocols, the total time to identify candidate loci for primer development from most eukaryotic species can be accomplished in as little as one week. *This information is based on (i.e., PLEASE CITE [using either style]): Glenn, T.C. and N.A. Schable. 2005. Isolating microsatellite DNA loci. Methods in Enzymology 395:202-222. or Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. Pp. 202-222 In: Methods in Enzymology 395, Molecular Evolution: Producing the Biochemical Data, Part B. (eds Zimmer EA, Roalson EH). Academic Press, San Diego, CA. Isolating Microsatellite DNA Loci p.2 Microsatellite DNA loci have become important sources of genetic information for a variety of purposes (Goldstein and Schlotterer, 1999; Webster and Reichart 2004).
    [Show full text]
  • Pspark® DNA Cloning System Manual for Cat
    pSpark® DNA cloning system Manual for cat. nº : C0001 (pSpark® I) C0002 (pSpark® II) C0003 (pSpark® III) C0004 (pSpark® IV) C0005 (pSpark® V) C0006 (pSpark® Done) Upon Receipt Store Kits at -20°C PRODUCT MANUAL Version 3.0 Last updated: December 2012 www.canvaxbiotech.com TABLE OF CONTENTS TABLE OF CONTENTS ii MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION DATE iii 1. INTRODUCTION 1 1.1 Principle and advantages 1 1.2 The family of pSpark® DNA cloning vectors 2 1.3 pSpark® DNA cloning vector maps 3 1.4 Specialized applications of the pSpark® DNA cloning systems 4 1.5 Additional materials required (but NOT supplied with kits unless otherwise stated)4 2. DETAILED PROTOCOL 5 2.1 Experimental outline 5 2.2 PCR 6 2.2.1 PCR Primers design 6 2.2.2 PCR Amplification 6 2.3 Ligation 8 2.3.1 Amount of insert needed for ligation into pSpark® DNA cloning systems. 8 2.3.2 Protocol for ligation using the pSpark® DNA cloning systems 10 2.3.3 Tips for cloning of long or problematic PCR products. 11 2.4 Transformation 12 2.4.1 General considerations about transformation into E coli 12 2.4.2 Standard protocol for transformation 13 2.4.3 Fast transformation protocol (Recommended alternative) 14 2.4.4 Transformation by electroporation protocol 16 2.5 Selection of recombinants 17 2.5.1 Expected results 17 2.6 Analysis of transformants 20 2.6.1 PCR directly from bacterial colonies (Colony PCR protocol) 20 2.6.2 Isolation of plasmid DNA 21 2.6.3 Sequencing 22 2.6.4 Long term storage of sequence-verified clones 22 3.
    [Show full text]