DOCSLIB.ORG

Molecular Cloning

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Now includes Recombinant Albumin Buffers Molecular Cloning TECHNICAL GUIDE be INSPIRED drive DISCOVERY stay GENUINE OVERVIEW TABLE OF CONTENTS 3 Online Tools 4–5 Cloning Workflow Comparison 6 DNA Assembly Molecular Cloning Overview 6 Overview Molecular cloning refers to the process by which recombinant DNA molecules are 6 Product Selection produced and transformed into a host organism, where they are replicated. A molecular 7 Golden Gate Assembly Kits cloning reaction is usually comprised of two components: 7 Optimization Tips 8 Technical Tips for Optimizing 1. The DNA fragment of interest to be replicated. Golden Gate Assembly Reactions 2. A vector/plasmid backbone that contains all the components for replication in the host. 9 NEBuilder® HiFi DNA Assembly 10 Protocol/Optimization Tips ® DNA of interest, such as a gene, regulatory element(s), operon, etc., is prepared for cloning 10 Gibson Assembly by either excising it out of the source DNA using restriction enzymes, copying it using 11 Cloning & Mutagenesis PCR, or assembling it from individual oligonucleotides. At the same time, a plasmid vector 11 NEB PCR Cloning Kit is prepared in a linear form using restriction enzymes (REs) or Polymerase Chain Reaction 12 Q5® Site-Directed Mutagenesis Kit (PCR). The plasmid is a small, circular piece of DNA that is replicated within the host and 12 Protocols/Optimization Tips exists separately from the host’s chromosomal or genomic DNA. By physically joining the 13–24 DNA Preparation DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest 13 Nucleic Acid Purification becomes part of the new recombinant plasmid and is replicated by the host. Plasmid vectors 13 Overview allow the DNA of interest to be copied easily in large amounts, and often provide the 14 cDNA Synthesis necessary control elements to be used to direct transcription and translation of the cloned 14 Overview DNA. As such, they have become the workhorse for many molecular methods such as 15 Restriction Enzyme Digestion 15 Overview protein expression, gene expression studies, and functional analysis of biomolecules. 15 Protocol/Optimization Tips 16–21 Performance Chart During the cloning process, the ends of the DNA of interest and the vector have to be 22 Activity in rCutSmart Buffer modified to make them compatible for joining through the action of a DNA ligase, 23 PCR/Amplification recombinase, or an in vivo DNA repair mechanism. These steps typically utilize enzymes 23 Overview such as nucleases, phosphatases, kinases and/or ligases. Many cloning methodologies and, 23 Product Selection more recently kits have been developed to simplify and standardize these processes. 24 Protocols/Optimization Tips 25–27 Common DNA End Modifications This technical guide will clarify the differences between the various cloning methods, 25 Overview identify NEB® products available for each method, and provide expert-tested protocols and 25 Phosphorylation FAQs to help you troubleshoot your experiments and Clone with Confidence®. 25 Protocol/Optimization Tips 25 Dephosphorylation 25 Product Selection 25 Protocol/Optimization Tips 26 Blunting/End-repair 26 Product Selection Visit CloneWithNEB.com 26 Protocol/Optimization Tips 27 A-tailing 27 Product Selection 27 Protocol 28 DNA Ligation 28 Vector and Insert Joining 28 Overview 28 Product Selection 29 Protocol/Optimization Tips 30 Transformation 30 Overview 30 Product Selection 30 Protocol/Optimization Tips 31 DNA Analysis 31 DNA Markers and Ladders 31 Overview 31 Product Selection • Technical tips and FAQs 32 Getting Started with Molecular Cloning 33–34 Traditional Cloning Quick Guide • Videos and animations 35–37 Troubleshooting Guide • Much more... 2 38–39 Ordering Information CLONING TOOLS Online Tools for Cloning Competitor Cross-Reference Tool NEBcutter® V3.0 Use this tool to select another company's competent cell Identify restriction sites within your DNA sequence product and find out which NEB strain is compatible. using NEBcutter. Choose between Type II and Choose either the product name or catalog number from commercially available Type III enzymes to digest your the available selection, and this tool will identify the DNA. NEBcutter V3.0 indicates cut frequency and recommended NEB product and its advantages. A link to the product methylation sensitivity. page where you can also order the product is provided. NEBioCalculator® DNA Sequences and Maps Tool NEBioCalculator is a collection of calculators and With the DNA Sequences and Maps Tool, find converters that are useful in planning bench experiments the nucleotide sequence files for commonly used in molecular biology laboratories. molecular biology tools, including plasmid, viral and bacteriophage vectors. NEBuilder® Assembly Tool Double Digest Finder NEBuilder Assembly Tool can be used to design Use this tool to guide your reaction buffer selection primers for your NEBuilder HiFi and Gibson Assembly when setting up double-digests, a common timesaving reaction, based on the entered fragment sequences and procedure. Choosing the right buffers will help you to the polymerase being used for amplification. avoid star activity and loss of product. PCR Selector Enzyme Finder Use this tool to help select the right DNA polymerase Use this tool to select restriction enzymes by name, for your PCR setup. Whether your amplicon is long, sequence, overhang or type. Enter your sequence using complex, GC-rich or present in a single copy, the PCR single letter code, and Enzyme Finder will identify the selection tool will identify the perfect DNA polymerase right enzyme for the job. for your reaction. Ligase Fidelity Tools REBASE® Visualize overhang ligation preferences, predict Use this tool as a guide to the ever-changing landscape high-fidelity junction sets, and split DNA sequences of restriction enzymes. REBASE, the Restriction Enzyme to facilitate the design of high-fidelity Golden Gate DataBASE, is a dynamic, curated database of restriction assemblies. enzymes and related proteins. NEB Golden Gate Assembly Tool Tm Calculator Use this tool to assist with in silico DNA construct design Use this tool when designing PCR reaction protocols for Golden Gate DNA assembly. It enables the accurate to help determine the optimal annealing temperature design of primers with appropriate Type IIS restriction for your amplicon. Simply input your DNA polymerase, sites and overlaps, quick import of sequences in many primer concentration and your primer sequence and the formats and export of the final assembly, primers and settings. The Tm Calculator will guide you to successful reaction conditions. latest version (v2.1) also incorporates ligase fidelity information. MOBILE APPS NEBaseChanger® NEBaseChanger can be used to design primers specific NEB Tools for iPhone®, iPad® or Android™ to the mutagenesis experiment you are performing NEB Tools brings New England Biolabs’ most popular web tools ® using the Q5 Site-Directed Mutagenesis Kit. This tool to your iPhone, iPad or Android devices. will also calculate a recommended custom annealing temperature based on the sequence of the primers by taking into • Use Enzyme Finder to select a restriction enzyme by category or recognition account any mismatches. sequence, or search by name to find information on any NEB enzyme. Sort your results so they make sense to you, then email them to your inbox or connect directly to www.neb.com. NEBcloner® • Use Double Digest Finder or NEBcloner to determine buffer and reaction conditions Use this tool to find the right products and protocols for experiments requiring two restriction enzymes. for each step (digestion, end modification, ligation ™ ® ™ and transformation) of your next traditional cloning When using either of these tools, look for rCutSmart , HF and Time-Saver experiment. Also, find other relevant tools and resources enzymes for the ultimate in convenience. NEB Tools enables quick and easy access to the most requested restriction enzyme information, and allows you to plan your to enable protocol optimization. experiments from anywhere. 3 WORKFLOW COMPARISON Cloning Workflow Comparison The figure below compares the steps for the various cloning methodologies, along with the time needed for each step in the workflows. INSERT PREPARATION VECTOR PREPARATION Multiple cloning site Starting Starting (MCS) Material Material RNA OR OR OR OR cDNA Synthesis Plasmid gDNA PCR products Annealed oligos cDNA Plasmid Traditional Cloning OR PCR Cloning OR Seamless Cloning OR LIC (Ligation OR Recombinational Restriction Enzyme OR PCR (RE Digestion & Ligation) (TA & Blunt-End) (Gene Assembly) Independent Cloning) (Gateway/Creator/Univector) (RE) Digestion RE Digestion PCR PCR PCR PCR RE Digestion PCR DNA DNA Preparation 60 min. (Standard) 90 min. 90 min. 90 min. 90 min. Processing 60 min. (Standard) 2 hr. 5–15 min. (Time-Saver) 5–15 min. (Time-Saver) Clean Up 15 min. P P P dsDNA P P P intermediate P OR OR OR OR Clean Up OR A A A A A A 15 min. P Dephosphorylation Clean Up Clean Up Clean Up Linear vector Blunting 15 min. 15 min. 15 min. (Optional) Dephosphorylation (Optional) T-addition DNA End Phosphorylation Cohesive-End Cohesive-End RE Digest DNA End 10–30 min. 10–30 min. 1.5 hr. Modifications (Optional) Formation by Formation by 60 min. (Standard) Modifications 30 min. 5´ → 3´ exo 3´ → 5´ exo 5–15 min. (Time-Saver) Clean Up 15 min OR P 30 min. 30 min. Gel & Column Purification 75 min. T dsDNA OR OR P T intermediate 2 P P P OR A + A Clean Up P P OR P 15 min. Gel and Column Site-Specific Purification Vector & Insert Recombination Linear vector, 75 min. Ligation Ligation Annealing Joining 60 min. ready for joining 15 min. Occurs simultaneously 30 min. Ligation with previous step Proteinase K Treatment Instant – 15 min. Recombination 10 min. Estimated total time 20 min. – 2 hr., 25 min. 3 hr., 45 min. sites 70 min.** T A A T OR OR Holding vector Assembled vector Endpoint vector Estimated total time* 1 hr., 20 min. – 3 hr. 2 hr. – 2 hr., 30 min. 2 hr., 15 min. 2 hr., 45 min.
Recommended publications
  • Improving DNA Plasmid Production in Escherichia Coli

    Improving DNA Plasmid Production in Escherichia Coli

    Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER (Under the Direction of Mark A. Eiteman) ABSTRACT The ability to produce large quantities of plasmid DNA is imperative for wide scale availability of DNA vaccines. Large scale, high yield production relies on the synergy between host strain, plasmid, medium and production scheme. Screening as many variables as quickly and cost effectively as possible is the goal. In this study, Escherichia coli strains were transformed with two plasmids and screened for plasmid yield in shake flasks in chemically defined medium supplemented with either glucose or glycerol. High yield candidates were grown in feed batch fermentations at two specific growth rates, µ = 0.14 h-1 and µ = 0.24 h-1. As predicted, high production in shake flasks was predictive of high production in fermentations. Using our media and process, we were able to reach volumetric yields of approximately 600 mg/L and specific yields of approximately 17.82 mg/g, regardless of growth rate. We were also able to increase productivity (mg/Lh) over 30%. INDEX WORDS: E. coli, fed-batch, gene therapy, plasmid production Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER B.S., Biological Engineering, University of Georgia, 1998 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2007 © 2007 Adam Singer All Rights Reserved Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER Major Professor: Mark A. Eiteman Committee: Elliot Altman Sidney Kushner Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2007 iv DEDICATION To my wife Dana and my daughter Sydney-Rose.

  • Regulation of ATP Levels in Escherichia Coli Using CRISPR

    Tao et al. Microb Cell Fact (2018) 17:147 https://doi.org/10.1186/s12934-018-0995-7 Microbial Cell Factories RESEARCH Open Access Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production Sha Tao, Ying Qian, Xin Wang, Weijia Cao, Weichao Ma, Kequan Chen* and Pingkai Ouyang Abstract Background: Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural favonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modifcation of ATP concentration in Escherichia coli was applied in efcient pinocembrin production. Results: In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference sys- tem was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efciency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a fnal pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains. Conclusions: Our results introduce a strategy to approach the efcient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condi- tion have been optimized for pinocembrin production.
  • Synthesis of a Contiguous 32-Kb Polyketide Synthase Gene Cluster

    Synthesis of a Contiguous 32-Kb Polyketide Synthase Gene Cluster

    Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster Sarah J. Kodumal, Kedar G. Patel, Ralph Reid, Hugo G. Menzella, Mark Welch, and Daniel V. Santi* Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545 Communicated by Robert M. Stroud, University of California, San Francisco, CA, September 17, 2004 (received for review July 19, 2004) To exploit the huge potential of whole-genome sequence infor- Our efforts to this end stemmed from a desire to develop mation, the ability to efficiently synthesize long, accurate DNA heterologous expression of large polyketide synthase (PKS) sequences is becoming increasingly important. An approach pro- genes in Escherichia coli. Type I modular PKS genes encode the posed toward this end involves the synthesis of Ϸ5-kb segments of giant enzymes (among the largest proteins known) that synthe- DNA, followed by their assembly into longer sequences by con- size polyketide natural products such as erythromycin, epothi- ventional cloning methods [Smith, H. O., Hutchinson, C. A., III, lone, and tacrolimus (12). These genes reside within the high Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, GϩC genomes of the actinomycete and myxobacterial groups of 15440–15445]. The major current impediment to the success of this prokaryotes and encode proteins with multiple sets, or modules, tactic is the difficulty of building the Ϸ5-kb components accurately, of active sites (domains). Each module catalyzes the assembly of efficiently, and rapidly from short synthetic oligonucleotide build- a specific two-carbon-unit component of the polyketide product. ing blocks.
  • Recombinant DNA and Elements Utilizing Recombinant DNA Such As Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology

    Recombinant DNA and Elements Utilizing Recombinant DNA Such As Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology

    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 by Amy B. Vento and David R. Gillum Office of Environmental Health and Safety University of New Hampshire June 3, 2002 Introduction Recombinant DNA (rDNA) has various definitions, ranging from very simple to strangely complex. The following are three examples of how recombinant DNA is defined: 1. A DNA molecule containing DNA originating from two or more sources. 2. DNA that has been artificially created. It is DNA from two or more sources that is incorporated into a single recombinant molecule. 3. According to the NIH guidelines, recombinant DNA are molecules constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or molecules that result from their replication. Description of rDNA Recombinant DNA, also known as in vitro recombination, is a technique involved in creating and purifying desired genes. Molecular cloning (i.e. gene cloning) involves creating recombinant DNA and introducing it into a host cell to be replicated. One of the basic strategies of molecular cloning is to move desired genes from a large, complex genome to a small, simple one. The process of in vitro recombination makes it possible to cut different strands of DNA, in vitro (outside the cell), with a restriction enzyme and join the DNA molecules together via complementary base pairing. Techniques Some of the molecular biology techniques utilized during recombinant DNA include: 1.
  • Restriction-Modification Gene Complexes As Selfish Gene Entities: Roles of a Regulatory System in Their Establishment, Maintenan

    Restriction-Modification Gene Complexes As Selfish Gene Entities: Roles of a Regulatory System in Their Establishment, Maintenan

    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6442–6447, May 1998 Microbiology Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion (programmed cell deathyepigeneticsyintragenomic conflictsyphage exclusionyplasmid) Y. NAKAYAMA AND I. KOBAYASHI* Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo 108-8639, Japan Communicated by Allan Campbell, Stanford University, Stanford, CA, March 6, 1998 (received for review May 31, 1997) ABSTRACT We have reported some type II restriction- cell has lost its RM gene complex, its descendants will contain modification (RM) gene complexes on plasmids resist displace- fewer and fewer molecules of the modification enzyme. Eventu- ment by an incompatible plasmid through postsegregational host ally, their capacity to modify the many sites needed to protect the killing. Such selfish behavior may have contributed to the spread newly replicated chromosomes from the remaining pool of restric- and maintenance of RM systems. Here we analyze the role of tion enzyme may become inadequate. Chromosomal DNA will regulatory genes (C), often found linked to RM gene complexes, then be cleaved at the unmodified sites, and the cells will be killed in their interaction with the host and the other RM gene com- (refs. 2–4; N. Handa, A. Ichige, K. Kusano, and I.K., unpublished plexes. We identified the C gene of EcoRV as a positive regulator results). This result is similar to the postsegregational host killing of restriction. A C mutation eliminated postsegregational killing mechanisms for maintenance of several plasmids (5). These RM by EcoRV.
  • Molecular Cloning of DNA Fragments Produced by Restriction

    Molecular Cloning of DNA Fragments Produced by Restriction

    Proc. Natl. Acad. Sci. USA Vol. 73, No. 5, pp. 1537-1541, May 1976 Biochemistry Molecular cloning of DNA fragments produced by restriction endonucleases SailI and BamI (DNA joining/plasmid/insertional inactivation of genes/Drosophila melanogaster) DEAN H. HAMER AND CHARLES A. THOMAS, JR. Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Bernard D. Dats, February 25,1976 ABSTRACT The highly specific restriction endonucleases cleaves various DNAs about once every 8 kb (kilobases), as SalI and BamI produce DNA fragments with complementary, compared to about once every 4 kb for EcoRI (16, 18). The cohesive termini that can be covalently joined by DNA ligase. resulting fragments have cohesive termini, and can be joined The Escherichia coli kanamycin resistance factor pML21 has to one another in head-to-tail, head-to-head, and one Sall site, at which DNA can be inserted without interfering probably with the expression of drug resistance or replication of the tail-to-tail orientation. Another highly specific restriction en- plasmid. A more convenient cloning vehicle can be made with donuclease that produces cohesive termini is BamI, from Ba- the tetracycline resistance factor pSC101, since insertion of cillus amyloliqefaciens H (G. Wilson and F. Young, personal DNA either at its single site for Sall or at that for BamI inacti- communication). We have shown that it cleaves D. melano- vates plasmid-specified drug resistance but not replication. To gaster DNA about once every 6 kb. This report describes the take advantage of this insertional inactivation, pSC101 was construction and verification of plasmid vehicles that allow one joined to a ColEl-ampicillin resistance plasmid having no Sall site, and to a ColEl-kanamycin resistance plasmid having no to clone and amplify potentially any DNA fragment produced BamI site.
  • Subcloning, Expression, and Enzymatic Study of PRMT5

    Subcloning, Expression, and Enzymatic Study of PRMT5

    Georgia State University ScholarWorks @ Georgia State University Biology Theses Department of Biology Summer 7-12-2010 Subcloning, Expression, and Enzymatic Study of PRMT5 Ran Guo Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/biology_theses Part of the Biology Commons Recommended Citation Guo, Ran, "Subcloning, Expression, and Enzymatic Study of PRMT5." Thesis, Georgia State University, 2010. https://scholarworks.gsu.edu/biology_theses/26 This Thesis is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Theses by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. SUBCLONING, EXPRESSION, AND ENZYMATIC STUDY OF PRMT5 by RAN GUO Under the Direction of Yujun George Zheng ABSTRACT Protein arginine methyltransferases (PRMTs) mediate the transfer of methyl groups to arginine residues in histone and non-histone proteins. PRMT5 is an important member of PRMTs which symmetrically dimethylates arginine 8 in histone H3 (H3R8) and arginine 3 in histone H4 (H4R3). PRMT5 was reported to inhibit some tumor suppressors in leukemia and lymphoma cells and regulate p53 gene, through affecting the promoter of p53. Through methylation of H4R3, PRMT5 can recruit DNA-methyltransferase 3A (DNMT3A) which regulates gene transcription. All the above suggest that PRMT5 has an important function of suppressing cell apoptosis and is a potential anticancer target. Currently, the enzymatic activities of PRMT5 are not clearly understood. In our study, we improved the protein expression methodology and greatly enhanced the yield and quality of the recombinant PRMT5.
  • Restriction Endonucleases

    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.
  • From Genes to Genomes

    From Genes to Genomes

    From Genes to Genomes Third Edition Concepts and Applications of DNA Technology Jeremy W. Dale, Malcolm von Schantz and Nick Plant University of Surrey, UK ©WILEY-BLACKWELL A John Wiley & Sons, Ltd., Publication Preface xiii 1 From Genes to Genomes 1 1.1 Introduction 1 1.2 Bask molecular biology 4 1.2.1 The DNA backbone 4 1.2.2 The base pairs 6 1.2.3 RNA structure 10 1.2.4 Nucleic acid synthesis 11 1.2.5 Coiling and supercoilin 11 1.3 What is a gene? 13 1.4 Information flow: gene expression 15 1.4.1 Transcription 16 1.4.2 Translation 19 1.5 Gene structure and organisation 20 1.5.1 Operons 20 1.5.2 Exons and introns 21 1.6 Refinements of the model 22 2 How to Clone a Gene 25 2.1 What is cloning? 25 2.2 Overview of the procedures 26 2.3 Extraction and purification of nucleic acids 29 2.3.1 Breaking up cells and tissues 29 2.3.2 Alkaline denaturation 31 2.3.3 Column purification 31 2.4 Detection and quantitation of nucleic acids 32 2.5 Gel electrophoresis 33 2.5.1 Analytical gel electrophoresis 33 2.5.2 Preparative gel electrophoresis 36 vi CONTENTS 2.6 Restriction endonudeases 36 2.6.1 Specificity 37 2.6.2 Sticky and blunt ends 40 2.7 Ligation 42 2.7.1 Optimising Ligation conditions 44 2.7.2 Preventing unwanted Ligation: alkaline phosphatase and double digests 46 2.7.3 Other ways of joining DNA fragments 48 2.8 Modification of restriction fragment ends 49 2.8.1 Linkers and adaptors 50 2.8.2 Homopolymer tailing 52 2.9 Plasmid vectors 53 2.9.1 Plasmid replication 54 2.9.2 Cloning sites 55 2.9.3 Selectable markers 57 2.9.4 Insertional inactivation
  • Experiment 2 Plasmid DNA Isolation, Restriction Digestion and Gel Electrophoresis

    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.
  • Gibson Assembly Cloning Guide, Second Edition

    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.
  • Supplementary Table S1. Table 1. List of Bacterial Strains Used in This Study Suppl

    Supplementary Table S1. Table 1. List of Bacterial Strains Used in This Study Suppl

    Supplementary Material Supplementary Tables: Supplementary Table S1. Table 1. List of bacterial strains used in this study Supplementary Table S2. List of plasmids used in this study Supplementary Table 3. List of primers used for mutagenesis of P. intermedia Supplementary Table 4. List of primers used for qRT-PCR analysis in P. intermedia Supplementary Table 5. List of the most highly upregulated genes in P. intermedia OxyR mutant Supplementary Table 6. List of the most highly downregulated genes in P. intermedia OxyR mutant Supplementary Table 7. List of the most highly upregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Table 8. List of the most highly downregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Figures: Supplementary Figure 1. Comparison of the genomic loci encoding OxyR in Prevotella species. Supplementary Figure 2. Distribution of SOD and glutathione peroxidase genes within the genus Prevotella. Supplementary Table S1. Bacterial strains Strain Description Source or reference P. intermedia V3147 Wild type OMA14 isolated from the (1) periodontal pocket of a Japanese patient with periodontitis V3203 OMA14 PIOMA14_I_0073(oxyR)::ermF This study E. coli XL-1 Blue Host strain for cloning Stratagene S17-1 RP-4-2-Tc::Mu aph::Tn7 recA, Smr (2) 1 Supplementary Table S2. Plasmids Plasmid Relevant property Source or reference pUC118 Takara pBSSK pNDR-Dual Clonetech pTCB Apr Tcr, E. coli-Bacteroides shuttle vector (3) plasmid pKD954 Contains the Porpyromonas gulae catalase (4)