DOCSLIB.ORG

Assignment 1 Dilutions and Concentrations Only Submit the Answers to One Digit After the Decimal, You Do Not Need to Show Your Calculations (1 Point/Question) 1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Assignment 1 Dilutions and Concentrations Only Submit the Answers to One Digit After the Decimal, You Do Not Need to Show Your Calculations (1 Point/Question) 1 Molecular Biology 2016 Assignment 1 Dilutions and Concentrations Only submit the answers to one digit after the decimal, you do not need to show your calculations (1 point/question) 1. You add water to 125 mL of 1.6 M LiCl to obtain 1.0 L of solution, what is the new concentration of LiCl? 2. In the previous problem, the solution contains how many parts of solute and of solvent? 3. You start with 2.5L of a KNO3 stock solution and wish to prepare 10.0 L of 1.5 M KNO3. What molarity would the potassium nitrate stock solution need to be if you were to use it all? 4. How many milliliters of 5.0 M copper sulfate solution must be added to 228 mL of water to achieve a 0.25 M copper sulfate solution? 5. 40.0 mL of 2.0 M Fe(NO3)3 is mixed with 2 mL of 5 M Fe(NO3) and 48 mL of water. What is the final molar concentration of Fe(NO3)? 6. You add 3.5 L of an HCl solution of unknown concentration to 2.0 L of 0.5 M HCl and obtain a solution with a final concentration of 1.5 M. What was the unknown concentration of the initial HCl solution? 7. You have a solution representing 126g/L of NaF (MW: 42g/mole). 180 mL of this solution is added to water to obtain a final volume of 1080 mL. What is the molarity of the resulting solution? 8. What is the molar concentration of chloride ions in a solution prepared by mixing 100.0 mL of 2.0 M KCl with 50.0 mL of a 1.50 M CaCl2 solution? 9. A solution is prepared by dissolving 54 g of AgNO3 in 156 mL of water. What is the percent concentration (m/m) of AgNO3 of this solution? 10. The A260nm of a DNA solution is 0.12. How much of this DNA solution and a 5.5X loading dye should you add to 15µL of water to obtain a sample which contains 30ng of DNA in 0.5X loading dye? (A260nm of 1.0 = 50 µg/mL DNA) Molecular Biology 2016 Restriction Enzymes & Restriction Mapping (3 points/question) 11. The nomenclature of restriction enzymes can provide useful information about the source of the enzyme. For example, EcoRI indicates that this enzyme was the first enzyme isolated from an E.coli strain “R”. From which bacteria was PvuII isolated from? 12. Define the following terms: Isoschizomer, neoschizomer, and isocaudomer. 13. Amongst the enzymes listed below, which if any, generate compatible ends to each other? (Ex. A and B) Enzyme Recognition Sequence Cut EcoRI 5'GAATTC 5'---G AATTC---3' BamHI 5'GGATCC 5'---G GATCC---3' HindIII 5'AAGCTT 5'---A AGCTT---3' TaqI 5'TCGA 5'---T CGA---3' NotI 5'GCGGCCGC 5'---GC GGCCGC---3' Sau3AI 5'GATC 5'--- GATC---3' PvuII 5'CAGCTG 5'---CAG CTG---3' KpnI 5'GGTACC 5'---GGTAC C---3' PstI 5'CTGCAG 5'---CTGCA G---3' SacI 5'GAGCTC 5'---GAGCT C---3' SalI 5'GTCGAC 5'---G TCGAC---3' ScaI 5'AGTACT 5'---AGT ACT---3' SpeI 5'ACTAGT 5'---A CTAGT---3' SphI 5'GCATGC 5'---GCATG C---3' StuI 5'AGGCCT 5'---AGG CCT---3' XbaI 5'TCTAGA 5'---T CTAGA---3' Molecular Biology 2016 14. The linear 12 Kbp DNA fragment shown below has cleavage sites for BamHI and EcoRI. The numbers indicate the distance in kilobases. Complete the table to indicate the fragment sizes which would be observed on an agarose gel following each of the indicated digests. Note, if different fragments of the same size are generated, the size should only be indicated once. (For example do not indicate 2Kbp and 2Kbp) B E B E 1 4 6 10 Enzyme digest Fragment sizes BamHI EcoRI BamHI + EcoRI 15. What fragment sizes could be generated from a BamHI partial digest? Only indicate the sizes of intermediate fragments which would not be obtained following a complete digest. (Fragments which contain one or more BamHI site which remains undigested) 16. Consider the results of the BamHI digest you indicated in the above table. Draw all possible maps, (including the one illusrated above) which could correspond to the results indicated for the BamHI digest alone. 17. It was determined that the enzyme XhoI cuts at 2.0Kbp on the map shown above. Indicate which BamHI fragment would be cut by XhoI and what sizes would be generated in each of the maps provided for question 16. (Ex. 4Kbp BamHI → 3 + 1 Kbp). 18. A complete digest with EcoRI + BamHI of 12µg of the above fragment was performed. Indicate the amount in µg of each of the fragments which would be obtained. Molecular Biology 2016 19. The circular 10Kbp DNA molecule shown below has cleavage sites for BamHI and EcoRI. Complete the table to indicate the fragment sizes which would be observed on an agarose gel following each of the indicated digests. Note, if different fragments of the same size are generated, the size should only be indicated once. (For example do not indicate 2Kbp and 2Kbp) 10/0 Kbp EcoRI BamHI 3 Kbp 6 Kbp BamHI Enzyme digest Fragment sizes BamHI EcoRI BamHI + EcoRI 20. What fragment sizes could be generated from a BamHI partial digest? Only indicate the sizes of intermediate fragments which would not be obtained following a complete digest. (Fragments which contain one or more BamHI site which remains undigested) 21. Consider that the EcoRI site is invariable. Is this the only possible map according to the results presented in the above table? If not draw another possible map. 22. It was determined that the enzyme XhoI cuts at 9Kbp on the map shown above. Indicate which BamHI fragment would be cut by XhoI and what sizes would be generated in each of the maps provided for questions 19 and 21. (Ex. 4Kbp BamHI → 3 + 1 Kbp). Molecular Biology 2016 23. An 8.9 kb circular plasmid is digested with three restriction enzymes, EcoRI, BamHI and HindIII, individually and in combination, and the resulting fragment sizes are determined by means of electrophoresis. The results are as follows: EcoRI 8.9 kb BamHI 6 kb. 2.9 kb HindIII 8.9 kb EcoRI + BamHI 6 kb, 2.4 kb, 0.5 kb EcoRI + HindIII 7.4 kb, 1.5 kb BamHI + HindIII 5 kb, 2.9 kb, 1 kb BamHI + EcoRI + HindIII 5 kb, 2.4 kb, 1 kb, 0.5 kb Draw a possible restriction map based on these results. Set the EcoRI site as the origin. 24. Obtain the picture of the agarose gel electrophoresis of digests of the plasmid pBR322. (The file can be found on the course’s web site under the rubric “Sequences>pBR322”. Based on the results obtained, answer the following questions: a. How many times did PvuII cut within the plasmid? b. How many times did HincII cut within the plasmid? c. How many times did HincII cut within the PvuII fragment? d. What are the distances between the PvuII and the HincII sites? Molecular Biology 2016 Lab exercise (4 points/question) Dilutions exercise with micropipettors (Pg. 13) 1. Indicate the absorbance readings obtained for each of the following solutions which you prepared in the first lab exercise. If you are submitting the assignment as a group of 2, indicate the average absorbance for both samples. a. A 1.5mM solution of compound “A”. b. A 0.36% (m/v) solution of compound “B”. c. A 6% (v/v) solution of solution I. d. A solution containing 0.5mg of compound “A” and 0.1% (v/v) of compound “B”. e. A solution representing the following ratio: solution I: solution II : water : 2:1:2 Determining DNA concentration: (Pg. 14) 2. Submit a table of the DNA concentration determinations experiment performed on page 14 of the lab manual. Your table should include the following information: Standard DNA concentrations (µg/mL), corresponding A260 readings, and A260 readings of each of the unknown diluted solutions of DNA you prepared. 3. Submit a graph representing the A260 readings Vs standard DNA concentrations. Include a line of best fit, the R coefficient, and the formula of the line. 4. Determine from your graph, what DNA concentration in µg/mL corresponds to an A260 of 1.0. 5. According to the constant determined in the previous question, what was the DNA concentration of the undiluted unknown DNA solution provided? Restriction digests & agarose gel electrophoresis (Pg. 15-17) 6. Submit a figure and an appropriate figure legend of the agarose gel described on page 17 of the lab manual. 7. Submit a standard curve of the molecular weight ladder (Migration distance Vs. Size in Kbp) 8. Submit a table of the restriction digests of the recombinant plasmid which includes the following information: Enzyme used, number of cuts, fragment sizes observed. 9. In a caption accompanying the table submitted, indicate the total size of the plasmid, the size of the vector, the size of the insert, and the restriction site (s) in which the insert was introduced in the vector. 10. Provide a figure which represents a possible restriction map of the insert within the multiple cloning site of pUC9. Your map should be linear and only include the insert within the multiple cloning site. (See directives on this course’s web site) Molecular Biology 2016 Bioinformatics 1 11. Submit a table with the following information with regards to each of the unknown genes from the first bioinformatics exercise.
Load more

Recommended publications
  • The Role of NF-Κb and C/EBP Factors During Pathogen-Mediated
    The role of NF-B and C/EBP factors during pathogen-mediated activation of bovine interleukin 8 and beta-defensin in mammary epithelial cells Inaugural dissertation for the academic degree Doctor rerum naturalium of Mathematisch-Naturwissenschaftlichen Fakultät Universität Rostock By Shuzhen Liu (M. Sc.), born on March-02-1972, in Shanxi Province, China From the Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere in Dummerstorf Rostock 2009 URN: urn:nbn:de:gbv:28-diss2009-0185-5 Dean: Prof. Dr. Hendrik Schubert Reviewers: 1. Prof. Dr. Hans-Martin Seyfert Research Unit molecular biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany 2. Prof. Dr. Dieter G. Weiss Division of animal Physiology, Institute of Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Strasse 3, 18059 Rostock, Germany 3. PD Dr. Ulrike Gimsa Research Unit Behavioural Physiology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Date of defense: October 19th, 2009 Table of Contents TABLE OF CONTENTS 1. INTRODUCTION..................................................................................................................1 1.1 Mastitis as a challenge in general immunology..................................................................1 1.2 Innate immunity of the bovine mammary gland ................................................................2 1.3 Toll-like receptors (TLRs): main receptors perceiving the pathogen
    [Show full text]
  • 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.
    [Show full text]
  • 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]
  • CLONING, EXPRESSION ANALYSIS, and TRANSFORMATION VECTOR CONSTRUCTION of DAM HOMOLOGS in PEACH and POPLAR Yuhui Xie Clemson University, [email protected]
    Clemson University TigerPrints All Theses Theses 1-2011 CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR Yuhui Xie Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Part of the Plant Biology Commons Recommended Citation Xie, Yuhui, "CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR" (2011). All Theses. 1146. https://tigerprints.clemson.edu/all_theses/1146 This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR _______________________________________________ A Thesis Presented to the Graduate School of Clemson University ________________________________________________ In Partial Fulfillment of the Requirement for the Degree Master of Science Plant and Environmental Sciences _________________________________________________ by Yuhui Xie August 2011 ________________________________________________ Accepted by: Dr. Douglas G. Bielenberg, Committee Chair Dr. Haiying Liang Dr. Hong Luo ABSTRACT Genetic fine mapping and sequencing of the EVG locus in peach [Prunus persica (L.) Batsch] identified six tandem arrayed Dormancy-Associated MADS-box (DAM) genes as candidates for regulating growth cessation and terminal bud formation in the non-dormant evergrowing (evg) mutant. Since the mutant is lacking expression of six genes in the mapped locus, further functional analysis is needed to narrow the list of gene candidates for the non-dormant evg phenotype. Here I report three sets of experiments designed to functionally test DAM genes in peach and their homologs in a model tree, hybrid poplar.
    [Show full text]
  • Enzymes in Cloning Part I
    ˹̀/˺̀/˺̊˼̊ ENZYMES IN CLONING PART I Dr.Sarookhani ˺ ˹̀/˺̀/˺̊˼̊ Cloning --aa definition •• From the Greek --klon,klon, a twig •• An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody) •• An individual grown from a single somatic cell of its parent & genetically identical to it •• Clone: a collection of molecules or cells, all identical to an original molecule or cell Dr.Sarookhani ˻ ˹̀/˺̀/˺̊˼̊ Different types of Cloning 1. Reproductive Cloning 2. Therapeutic Cloning 3. Recombinant DNA Technology or DNA Cloning Dr.Sarookhani ˼ ˹̀/˺̀/˺̊˼̊ DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest. Dr.Sarookhani ̊ ˹̀/˺̀/˺̊˼̊ What is genetic engineering • Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. • Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all. Dr.Sarookhani ̋ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̌ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̀ ˹̀/˺̀/˺̊˼̊ Genomic Library Dr.Sarookhani ́ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̂ ˹̀/˺̀/˺̊˼̊ Basic steps in genetic engineering 1.
    [Show full text]
  • How to Clone Ref V1-2
    A primer on cloning in E. coli Michael Nonet Department of Neuroscience Washington University Medical School St Louis, MO 63110 Draft 1.2.1 Nov. 10, 2019 Things to add: gel electrophoresis methods basic bacteriology methods Index Overview E. coli vectors! 6 Types of E. coli vectors! 6 Components of typical plasmid vectors! 6 Origin of replication! 6 Antibiotic resistance genes! 7 Master Cloning Sites! 7 LacZ"! 7 ccdB ! 8 Design of DNA constructs! 9 Choice of vector! 9 Source of insert! 9 Plasmids! 9 Genomic or cDNA! 9 Synthetic DNA! 10 Overview of different approaches to creating clones! 10 Restriction enzyme based cloning! 10 Single vs. double cut method! 11 Blunt vs “sticky” restriction sites! 11 Typical methodology for restriction enzyme cloning! 11 Golden Gate style cloning! 12 Typical methodology for Golden Gate cloning! 12 Gateway cloning! 13 Typical methodology for Gateway cloning! 14 Gibson assembly cloning! 15 Typical procedure for Gibson assembly! 15 Site directed mutagenesis! 16 PCR overlap approach! 16 DpnI mediated site directed mutagenesis! 16 Typical procedure for site directed mutagenesis! 17 Detailed methodology for all five methods Restriction enzyme cloning! 18 Designing the cloning strategy ! 18 Determining which vector to use! 18 Designing the insert fragment! 19 Designing primers! 19 Vector preparation! 19 Insert preparation! 19 Detailed protocol! 20 Step 1: Design oligonucleotides to amplify the product of interest! 20 Step 2: PCR amplification of insert DNA! 20 Step 3: Clean-up purification of PCR product! 21 Step 4: Digestion of products! 21 Step 5: Gel purification of products! 22 Step 6: Ligation! 22 Step 7: Transformation of E.
    [Show full text]
  • Cyclic Digestion and Ligation-Mediated PCR Used For
    www.nature.com/scientificreports OPEN Cyclic Digestion and Ligation- Mediated PCR Used for Flanking Sequence Walking Dong Yu1,2,5, Tianshun Zhou2,4,5, Xuewu Sun2,3, Zhizhong Sun2, Xiabing Sheng1,2, Yanning Tan2, Ling Liu2,4, Ning Ouyang2,4, Ke Xu2, Kaibing Shi2, Guilong Yuan2, Jia Ding2, Meijuan Duan3* & Dingyang Yuan1,2,3,4* Ligation-mediated PCR (LM-PCR) is a classical method for isolating fanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and fexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 diferent adapters at low cost. Using this method, the fanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efcient and fexible method for identifying the fanking sequences in transgenic rice and Arabidopsis thaliana. Identifcation of fanking sequences has ofen been employed to determine the location of T-DNA insertion in genomic DNA. Methods to obtain fanking sequencea include inverse PCR1, randomly primed PCR2–5, and ligation-mediated PCR6–8. Inverse PCR, the earliest fanking cloning technique, has a low efciency and is limited by the rate of self-ligation and amplifcation range of DNA polymerases9.
    [Show full text]
  • The Cloning Guide the First Step Towards a Succesful Igem Project Igem Bonn NRP-UEA Igem
    The Cloning Guide The first step towards a succesful iGEM Project iGEM Bonn NRP-UEA iGEM iGEM Manchester-Graz iGEM Evry iGEM Vanderbilt iGEM Minnesota Carnegie Mellon iGEM iGEM Sydney UIUC iGEM iGEM York iGEM Toulouse iGEM Pasteur UCLA iGEM iGEM Stockholm Preface The cloning guide which is lying before you or on your desktop is a document brought to you by iGEM TU Eindhoven in collaboration with numerous iGEM (International Genetically Engi- neered Machine) teams during iGEM 2015. An important part in the iGEM competition is the collabration of your own team with other teams. These collaborations are fully in line with the dedications of the iGEM Foundation on education and competition, the advancement of syn- thetic biology, and the development of an open community and collaboration. Collaborations between groups of (undergrad and overgrad) students can lead to nice products, as we have tried to provide one for you in 2015. In order to compile a cloning guide, several iGEM teams from all over the world have been con- tacted to cooperate on this. Finally fifiteen teams contributed in a fantastic way and a cloning guide consisting of the basics about nine different cloning methods and experiences of teams working with them has been realized. Without the help of all collaborating teams this guide could never have been realized, so we will thank all teams in advance. This guide may be of great help when new iGEM teams (edition 2016 and later) are at the point of designing their project. How to assemble the construct for you project, is an important choice which is possibly somewhat easier to make after reading this guide.
    [Show full text]
  • Bpuami: a Novel Saci Neoschizomer from Bacillus Pumilus Discovered in an Isolate from Amazon Basin, Recognizing 5'-Gag↓Ctc-3'
    Brazilian Journal of Microbiology (2006) 37:96-100 ISSN 1517-8382 BPUAMI: A NOVEL SACI NEOSCHIZOMER FROM BACILLUS PUMILUS DISCOVERED IN AN ISOLATE FROM AMAZON BASIN, RECOGNIZING 5'-GAG↓CTC-3' Jocelei M. Chies1,2,*; Ana C. de O. Dias1; Hélio M. M. Maia3; Spartaco Astolfi-Filho2,3 1Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, RS, Brasil; 2Curso de Pós-Graduação em Biologia Molecular, Universidade de Brasília, Brasília, DF, Brasil; 3Centro de Apoio Multidisciplinar, Universidade Federal do Amazonas, Manaus, AM, Brasil Submitted: June 27, 2005; Returned to authors for corrections: November 16, 2005; Approved: January 12, 2006 ABSTRACT A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of “nonspecific nucleases.” Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG↓CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus. Key words: type II restriction endonuclease, BpuAmI, SacI, neoschizomers, Bacillus pumilus INTRODUCTION thousands of taxonomically diverse bacteria for enzymes with new characteristics. However, to our knowledge, there has Restriction endonucleases are enzymes which recognize not been any report to date of an isoschizomer and/or short DNA sequences and cleave DNA in both strands.
    [Show full text]
  • 1. Restriction Endonucleases (® Fermentas 2006)
    We guarantee that these products are Introduction ..........................................................................................................2 free of contaminating activities. Restriction Endonucleases Guide Our stringent quality control with the most FastDigest™ Restriction Endonucleases .........................................................2 Fermentas Restriction Endonucleases............................................................3 advanced tests guarantees you pure 1 Alphabetic List of Commercially Available Restriction Endonucleases.........6 products for your experiments. ISO9001 Recognition Specifi cities...............................................................................16 and ISO14001 is your assurance of con- Commercial Restriction Enzymes Sorted by the Type of DNA Ends Generated sistency and lot-to-lot reproducibility. Enzymes Generating 5’-protruding Ends.......................................................18 Enzymes Generating 3’-protruding Ends.......................................................18 ™ PureExtreme Quality will provide the Enzymes Generating Blunt Ends...................................................................19 performance you need for your most Enzymes Cleaving DNA on the Both Sides of Their Recognition Sequences.....19 demanding experiments. PureExtreme™ Quality PureExtreme™ Quality Guarantee..................................................................20 Activity Assay.................................................................................................20
    [Show full text]
  • A Short History of the Restriction Enzymes Wil A
    Published online 18 October 2013 Nucleic Acids Research, 2014, Vol. 42, No. 1 3–19 doi:10.1093/nar/gkt990 NAR Breakthrough Article SURVEY AND SUMMARY Highlights of the DNA cutters: a short history of the restriction enzymes Wil A. M. Loenen1,*, David T. F. Dryden2,*, Elisabeth A. Raleigh3,*, Geoffrey G. Wilson3,* and Noreen E. Murrayy 1Leiden University Medical Center, Leiden, the Netherlands, 2EaStChemSchool of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK and 3New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA Received August 14, 2013; Revised September 24, 2013; Accepted October 2, 2013 ABSTRACT Type II REases represent the largest group of characterized enzymes owing to their usefulness as tools for recombinant In the early 1950’s, ‘host-controlled variation in DNA technology, and they have been studied extensively. bacterial viruses’ was reported as a non-hereditary Over 300 Type II REases, with >200 different sequence- phenomenon: one cycle of viral growth on certain specificities, are commercially available. Far fewer Type I, bacterial hosts affected the ability of progeny virus III and IV enzymes have been characterized, but putative to grow on other hosts by either restricting or examples are being identified daily through bioinformatic enlarging their host range. Unlike mutation, this analysis of sequenced genomes (Table 1). change was reversible, and one cycle of growth in Here we present a non-specialists perspective on import- the previous host returned the virus to its original ant events in the discovery and understanding of REases. form. These simple observations heralded the dis- Studies of these enzymes have generated a wealth of covery of the endonuclease and methyltransferase information regarding DNA–protein interactions and catalysis, protein family relationships, control of restric- activities of what are now termed Type I, II, III and tion activity and plasticity of protein domains, as well IV DNA restriction-modification systems.
    [Show full text]
  • Conventional Nzytech Restriction Enzymes: Activity Guide
    Conventional NZYTech Restriction Enzymes: Activity Guide Enzyme Activity in NZYTech buffers (%) Buffer Buffer for Restriction site Methylation Reaction Enzyme provided for double Isoschizomers (5’ →→→3’) A (green) B (blue) C (red) U (white) sensitive Conditions 100% activity digestion AscI GG ↓↓↓CGCGCC NZYBuffer C 80 100 100 100 NZYBuffer U * PalAI, SgsI (MB231) BamHI G↓↓↓GATCC NZYBuffer B 40 100 (<20) 100 NZYBuffer U * No None (MB064) BglII A↓↓↓GATCT NZYBuffer A 100 (100) (60) 100 NZYBuffer U * No None (MB065) CciNI (NotI) GC ↓↓↓GGCCGC NZYBuffer A 100 80 60 100 NZYBuffer U * NotI (MB153) DdeI C↓↓↓TNAG NZYBuffer U 25 25 25 100 NZYBuffer U No BstDEI, HpyF3I (MB236) MalI, BfuCI ♦♦♦, Bsp143I ♦♦♦, BstENII ♦♦♦, DpnI G(mA)↓↓↓TC NZYBuffer C 70 50 100 100 NZYBuffer U * (MB078) BstKTI ♦♦♦, BstMBI ♦♦♦, DpnII ♦♦♦, Kzo9I ♦♦♦, NdeII ♦♦♦ BfuCI, Bsp143I, BssMI, BstKTI, BstMBI, DpnII ↓↓↓GATC NZYBuffer C 80 80 100 100 NZYBuffer U * (MB233) Kzo9I, MboI, NdeII, Sau3AI EcoRI G↓↓↓AATTC NZYBuffer A 100 (120) (80) 100 NZYBuffer U * FunII (MB067) EcoRV GAT ↓↓↓ATC NZYBuffer A 100 0 25-50 100 NZYBuffer U * Eco32I (MB068) HindIII A↓↓↓AGCTT NZYBuffer B <20 100 (100) 100 NZYBuffer U * No None (MB070) HinfI G↓↓↓ANTC NZYBuffer C (90) (90) 100 100 NZYBuffer U * None (MB239) HpaI GTT ↓↓↓AAC NZYBuffer B 20 100 (80) 100 NZYBuffer U * KspAI (MB071) KpnI GGTAC ↓↓↓C NZYBuffer C 50 (80) 100 100 NZYBuffer U * No Acc65I ♦♦♦, Asp718I ♦♦♦ (MB072) BfuCI, Bsp143I, BstENII, BstMBI, DpnII, MboI ↓↓↓GATC NZYBuffer C 100 100 100 100 NZYBuffer U * (MB241) Kzo9II, NdeII, BstKTI ♦♦♦ MluI
    [Show full text]