Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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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. -
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 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. -
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. -
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 -
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. -
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. -
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). -
Restriction Endonucleases
Restriction Endonucleases TECHNICAL GUIDE UPDATE 2017/18 be INSPIRED drive DISCOVERY stay GENUINE RESTRICTION ENZYMES FROM NEB Cut Smarter with Restriction Enzymes from NEB® Looking to bring CONVENIENCE to your workflow? Simplify Reaction Setup and Double Activity of DNA Modifying Enzymes in CutSmart Buffer: Digestion with CutSmart® Buffer Clone Smarter! Activity Enzyme Required Supplements Over 210 restriction enzymes are 100% active in a single buffer, in CutSmart Phosphatases: CutSmart Buffer, making it significantly easier to set up your Alkaline Phosphatase (CIP) + + + double digest reactions. Since CutSmart Buffer includes BSA, there Antarctic Phosphatase + + + Requires Zn2+ Quick CIP + + + are fewer tubes and pipetting steps to worry about. Additionally, Shrimp Alkaline Phosphatase (rSAP) + + + many DNA modifying enzymes are 100% active in CutSmart Ligases: T4 DNA Ligase + + + Requires ATP Buffer, eliminating the need for subsequent purification. E. coli DNA Ligase + + + Requires NAD T3 DNA Ligase + + + Requires ATP + PEG For more information, visit www.NEBCutSmart.com T7 DNA Ligase + + + Requires ATP + PEG Polymerases: T4 DNA Polymerase + + + DNA Polymerase I, Large (Klenow) Frag. + + + DNA Polymerase I + + + DNA Polymerase Klenow Exo– + + + Bst DNA Polymerase + + + ™ phi29 DNA Polymerase + + + Speed up Digestions with Time-Saver T7 DNA Polymerase (unmodified) + + + Qualified Restriction Enzymes Transferases/Kinases: T4 Polynucleotide Kinase + + + Requires ATP + DTT T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT > 190 of our restriction enzymes are able to digest DNA in CpG Methyltransferase (M. SssI) + + + 5–15 minutes, and can safely be used overnight with no loss of GpC Methyltransferase (M. CviPI) + Requires DTT T4 Phage β-glucosyltransferase + + + sample. For added convenience and flexibility, most of these are Nucleases, other: supplied with CutSmart Buffer. -
![Restriction Digest and Ligation] 25/09/2018 Igem Tuebingen 2018](https://docslib.b-cdn.net/cover/4888/restriction-digest-and-ligation-25-09-2018-igem-tuebingen-2018-1634888.webp)
Restriction Digest and Ligation] 25/09/2018 Igem Tuebingen 2018
[Restriction Digest and Ligation] 25/09/2018 iGEM Tuebingen 2018 Restriction Digest and Ligation The restriction digest and ligation protocol is used to transfer DNA fragments from one plasmid to another, as long as the DNA pieces have matching restriction sites. The restriction enzymes digest the DNA at the corresponding restriction sites, which results in complementary ends of the target plasmid and the insert. The ligase finally adds together target plasmid and insert. For this protocol the materials of NEB were used. Restriction Digest Materials - Restriction Endonucleases (NEB) - Restriction Digest Buffer (e.g. CutSmartTM Buffer) - Plasmid DNA Procedure - Prepare a 25 ul reaction: Component Amount Restriction Enonuclease 1 ul for each used enzyme CutSmartTM Buffer 3 ul Nuclease-free water Up to 25 ul - Incubation for 1h at 37°C (or at different temperature, dependent on enzyme) - Heat inactivate restriction enzymes at 65°C to 80°C (dependent on restriction enzyme) for 20 min Ligation Materials - T4 DNA Ligase Buffer (10x) - T4 DNA Ligase - Vector DNA - Insert DNA - Nuclease-free water Procedure For the protocol it is indispensable to know the sizes of the target vector and the insert. Normally a molar ratio of vector to insert of 1:3 is used for cohesive end ligations. A higher molar ratio of 1:4 or 1:5 can be used for ligations of DNA fragements with blunt ends - Prepare a 20 ul reaction (T4 DNA Ligase should be added last) 1 [Restriction Digest and Ligation] 25/09/2018 iGEM Tuebingen 2018 Component Amount T4 DNA Ligase Buffer (10x) 2 ul Vector DNA Dependent on vector size, e.g. -
DNA Molecular Weight Marker VIII (19 – 1114 Bp) Pucbm21 DNA × Hpa II* Digested Pucbm21 DNA × Dra I* + Hind III* Digested
For life science research only. Not for use in diagnostic procedures. DNA Molecular Weight Marker VIII (19 – 1114 bp) pUCBM21 DNA × Hpa II* digested pUCBM21 DNA × Dra I* + Hind III* digested ־ Cat. No. 11 336 045 001 50 g 1 A260 unit y Version 19 50 g (200 l) for 50 gel lanes Content version: September 2018 Store at Ϫ15 to Ϫ25°C 1. Product Overview Content Ready-to-use solution in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 Concentration 250 g/ml Size Distribution Fragment mixture prepared by cleavage of pUCBM21 DNA with restriction endonucleases Hpa II * and Dra I* plus Hind III*. The mixture contains 18 DNA fragments with the following base pair lengths (1 base pair = 660 daltons): 1114, 900, 692, 501, 489, 404, 320, 242, 190, 147, 124, 110, 67, 37, 34, (2×), 26, 19 (determined by computer analysis of the pUCBM21 sequence). Application Molecular weight marker for the size determination of DNA in agarose gels. Properties After gel electrophoresis of 1 g of the fragment mixture in a 2% Aga- rose MP* gel 13 bands are visible. The 501 bp and 489 bp fragments as well as the fragments 37–19 bp run as one band. Typical Analysis Fig. 1: Separation of 1 g DNA Molecular Weight Marker VIII on a The DNA fragment mixture shows the typical pattern of 13 bands in 1.8% agarose gel. Ethidiumbromide stain. agarose gel electrophoresis.. 2. Supplementary Information Storage/Stability The unopened vial is stable at Ϫ15 to Ϫ25°C until the expiration date Changes to Previous Version printed on the label. -
Gel Electrophoresis Applications Gel Electrophoresis Applications
Restriction Enzymes (also known as restriction endonucleases) What are restriction enzymes? • An enzyme that cuts DNA at specific nucleotide sequences known as restriction sites • Naturally found in bacteria and have evolved to provide a defense mechanism against invading viruses • In bacteria, restriction enzymes selectively cut up foreign DNA in a process called restriction Restriction Enzymes in Bacteria Restriction Enzymes • Cut both strands of the DNA double helix • Over 3000 enzymes have been identified • More than 600 available commercially • Routinely used for DNA modification and manipulation in laboratories (“molecular scissors”) Restriction Sites • Also known as recognition sites • Generally genetic palindromic sequences • A palindromic sequence in DNA one in which the 5’ to 3’ base pair sequence is identical on both strands • Usually a 4 or 6 base pair sequence Restriction Sites • The enzymes scan DNA sequences, find a very specific set of nucleotides and make a cut Hae III • HaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases - GGCC 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ Hae III • Once the recognition site was found HaeIII will go to work cutting (cleaving) the DNA Blunt Ends versus Sticky Ends • Hae III produces “blunt ends” when cleaving DNA • Other enzymes produce “sticky ends” blunt end sticky end Restriction Enzyme Names • Named after the type of bacteria in which the enzyme is found and the order in which the restriction enzyme was identified and isolated EcoRI for example R strain of E.coli bacteria I as it is was the first E. coli restriction enzyme to be discovered.