DOCSLIB.ORG

PCR Reagents

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
PCR Reagents AMPLIFICATION PCR Reagents + How do you ensure optimal amplification results? + How do you choose the best PCR enzyme for your application? + How can you improve cDNA synthesis? 0>0 PCR REAGENTS + We have the best PCR reagent for your application. Our expertise in enzyme engineering has produced one of the broadest portfolios of high-fidelity PCR enzymes available today. This expertise has translated to advances in high-fidelity reverse transcription PCR. Whether you need the highest accuracy available, large product yields, or sensitive cDNA synthesis, trust our high-performance enzymes to answer your amplification challenges. CONTENTS: PCR Enzymes & Master Mixes RT-PCR Kits & Reagents 6 Ultra-High Fidelity PCR 13 RT-PCR 7 High Fidelity for TA/UA Cloning 8 High Fidelity for Blunt-End Cloning PCR-Related Kits & Reagents 9 High Sensitivity & High Yield 14 Nucleotides 10 GC-Rich/Complex Targets 14 PCR Purification Kits 11 Long PCR 14 PCR Cloning Kits 12 Routine PCR 15 Ordering Information PCR ENZYMES Ultra-High Fidelity High Fidelity High Fidelity High Fidelity Format (Cloning & (Contamination (TA/UA Cloning) (Blunt Cloning) Mutagenesis) Control) ™ ® ® PfuUltraa™ Easy-A PfuTurboo PfuTurboo Cx Hotstart Enzyme Hotstart DNA High Fidelity PCR Hotstart DNA Hotstart DNA Polymerase oning Enzym Polymerase Polymerase PfuUltraa™ PfuTurbo® Standard Enzyme High-Fidelity DNA Polymerase DNA Polymerase PfuUltraa™ Easy-A™ PfuTurboo® Master Mix Hotstart PCR High Fidelity PCR Hotstart PCR Master Mix Master Mix Master Mix GC-Rich/ High Sensitivity/ Format Complex Long PCR Routine PCR High Yield Templates PicoMaxx™ Herculase® Hotstart Enzyme High Fidelity Hotstart DNA PCR System Polymerase Herculase® EXL™ Taq2000 ™ Standard Enzyme nhanced DN DNA Polymerase DNA Polymerase Polymerase PicoMaxx™ Herculase® Master Mix High Fidelity Hotstart PCR PCR Master Mix Master Mix RT-PCR KITS & REAGENTS Format High Yield/ High Sensitivity Fidelity StrataScript™ cDNA Synthesis First Strand Synthesis Kit StrataScript™ One-Tube RT-PCR One-Tube RT-PCR Kit with Easy-A™ PCR Cloning Enzyme StrataScript™ Two-Tube RT-P Two-Tube RT-PCR Kit with PicoMaxx™ PCR System PfuUltra™ DNA Polymerase www.stratagene.com 4>5 PCR REAGENTS The Importance of High Fidelity High-fidelity PCR enzymes are valuable for minimizing the introduction of amplification errors in products that will be cloned, sequenced, and expressed. Significant time and effort can be saved by employing high-fidelity amplification procedures that eliminate the need for downstream error-correction steps and minimize the number of clones that must be sequenced in order to obtain error-free constructs or accurate consensus sequences. Moreover, the use of high-fidelity amplification conditions is essential when analyzing small amounts of template DNA or rare molecules in heterogeneous populations. Amplifications employing small amounts of template DNA are especially prone to high mutant frequencies due to PCR-generated errors in early cycles1, 2. The ArchaeMaxx® Factor Advantage Because proofreading PCR enzymes can be finicky, we have improved high-fidelity PCR to be more reliable. Our ArchaeMaxx® Polymerase-Enhancing Factor, found exclusively in our high-fidelity DNA polymerases, improves overall PCR performance by eliminating the inhibition of proofreading enzymes caused by incorporation of dUTP, which results from deamination of dCTP during PCR3. Our PCR enzymes formulated with the ArchaeMaxx factor produce higher yields and exhibit greater target length capability. Superior Reverse Transcription Our expertise in enzyme engineering also extends to reverse transcriptases. Both MMLV and AMV reverse transcriptases possess substantial RNase H activity that degrades RNA molecules and limits full-length cDNA synthesis and yield. For this reason, RTs that lack RNase H activity are far superior to MMLV and other RNAse H+ RTs. Our StrataScript™ Reverse Transcriptase contains a point mutation that effectively abolishes RNase H activi- ty, allowing more efficient production of full-length cDNA than RNase H+ RTs. Comparison of Our PCR Enzymes PCR EnzymeAccuracy Target Blunt or ArchaeMaxx® Hot Start Master Mix vs. Taq Length 3'-A Ends Advantage Available Available 0-17 kb (genomic) PfuUltra™ High Fidelity DNA Polymerase 18x Blunt +++ 0-15 kb (vector) 0-19 kb (genomic) PfuTurbo® DNA Polymerase 6x Blunt +++ 0-15 kb (vector) ® PfuTurbo Cx DNA Polymerase 6x 0-10 kb Blunt + Pfu DNA Polymerase, cloned or native 6x 0-5 kb Blunt Easy-A™ High Fidelity PCR Cloning Enzyme 6x 0-5 kb 3'-A +++ Herculase® DNA Polymerase 3x 0-37 kb Mixed +++ 20-37 kb (genomic) EXL™ DNA Polymerase 3x Mixed + 20-50 kb (vector) PicoMaxx™ High Fidelity PCR System 2x 0-10 kb Mixed +++ SureStart® Taq DNA Polymerase 1x 0-5 kb 3'-A + Taq2000 ™ DNA Polymerase 1x 0-4 kb 3'-A Comparison of Our RT-PCR Kits & Reagents RT-PCR Reagent Target RNase H cDNA One-Tube Two-Tube Length Deficient Synthesis RT-PCR RT-PCR StrataScript™ First Strand Synthesis Kit 0-9 kb ++ StrataScript™ First-Strand Synthesis Kit 0-9 kb ++ + plus PicoMaxx™ High Fidelity PCR System StrataScript™ One-Tube RT-PCR Kit 0-5 kb ++ with Easy-A™ PCR Cloning Enzyme StrataScript™ Reverse Transcriptase 0-9 kb ++ StrataScript™ Two-Tube Kit 0-9 kb ++ + with PfuUltra™ DNA Polymerase www.stratagene.com 6>7 PCR REAGENTS > PCR ENZYMES & MASTER MIXES Ultra-High Fidelity PCR The Most Accurate PCR Enzyme Our continuing efforts to improve the fidelity of PCR have resulted in PfuUltra™ High-Fidelity DNA Polymerase, the most accurate PCR enzyme commercially available. PfuUltra DNA poly- merase is ideal for PCR cloning, site-directed mutagenesis, and anywhere sequence accuracy is critical for downstream applications. Enhanced Performance In addition to ultra-high fidelity, PfuUltra high-fidelity DNA PCR Enzyme Accuracy Target Blunt or ArchaeMaxx® Hot Start Master Mix vs. Taq Length 3'-A Ends Advantage Available Available polymerase exhibits superior PCR performance compared to PfuUltra™ 0-17 kb (genomic) most PCR proofreading enzymes. The addition of our ArchaeMaxx High Fidelity 18x Blunt +++ 0-15 kb (vector) DNA Polymerase polymerase-enhancing factor promotes higher yields, shorter extension times, and greater target length capability (Figure 2). High Specificity Hotstart Version Highest Fidelity Available The PfuUltra™ Hotstart DNA Polymeraseø,‡ is an antibody-mediated ™ ø,‡ The PfuUltra high-fidelity DNA polymerase contains a hotstart formulation of PfuUltra high fidelity DNA polymerase ***,‡ genetically engineered mutant of Pfu DNA polymerase that that provides higher specificity and reduced background. delivers 300% greater accuracy. A validated and referenced 4 fidelity assay demonstrates that PfuUltra high-fidelity DNA poly- Convenient Master Mix Formulation merase exhibits an average error rate three-fold lower than Pfu For ultra-high fidelity with added convenience and increased DNA polymerase and 18-fold lower than Taq DNA polymerase, throughput, choose PfuUltra™ Hotstart PCR Master Mix ø,‡. making it the most accurate PCR enzyme available (Figure 1). This 2x formulation contains PfuUltra hotstart DNA polymerase, buffer, magnesium, and dNTPs all in one tube for faster setup and more consistent results. DNA Polymerases ACCURACY x 105 M 0.97 1.5 3.5 6 9 12 17 kb 0 5 10 15 20 25 PfuUltra™ DNA Polymerase ™ ® PfuTurbo and PfuTurbo Cx DNA Polymerases Tgo DNA Polymerase Herculase® DNA Polymerase Pfx/KOD DNA Polymerase Expand™ High Fidelity Advantage-HF™ Polymerase Taq DNA Polymerase Figure 1 Figure 2 THE MOST ACCURATE PCR ENZYME AVAILABLE AMPLIFIES A WIDE RANGE OF TARGETS PfuUltra™ High-Fidelity DNA Polymerase provides the greatest accuracy of any commercially PfuUltra™ High-Fidelity DNA Polymerase amplifies genomic available enzyme1. Fidelity was measured using our validated and referenced fidelity assay4. DNA targets up to 17 kb. Accuracy = 1/Error Rate. High Fidelity for TA/UA Cloning Unique Proofreading Formulation Adds A-Overhangs TA or UA cloning requires PCR products with 3'-A overhangs. We created the Easy-A™ High-Fidelity PCR Cloning Enzyme to automatically add A-overhangs for quick and easy high-fidelity cloning into TA/UA vectors. Combines Cloning Efficiency with High Accuracy The Easy-A™ high-fidelity PCR cloning enzyme ,‡,† is the only PCR Enzyme Accuracy Target Blunt or ArchaeMaxx® Hot Start Master Mix proofreading DNA polymerase formulation to deliver high- vs. Taq Length 3'-A Ends Advantage Format Available throughput TA or UA cloning. PCR products amplified with the Easy-A™ High Fidelity PCR 6x0-5 kb 3'-A +++ Easy-A PCR cloning enzyme can be cloned directly to T- or Cloning Enzyme U-modified vectors with high cloning efficiency and throughput (Figure 3). But unlike Taq DNA polymerase, the Easy-A PCR cloning enzyme exhibits proofreading activity that provides the accuracy level of Pfu DNA polymerase. High Specificity Hotstart Formulation The Easy-A high fidelity PCR cloning enzyme is provided as Fidelity Without Extra Work an antibody-mediated hotstart formulation, providing higher Adapting blunt-ended fragments amplified with proofreading specificity and reduced background. enzymes requires post-PCR addition of 3'-A overhangs prior to the cloning step. This additional step includes a secondary Convenient Master Mix Formulation ™ incubation with Taq DNA polymerase and requires the opening For the highest throughput in TA or UA cloning, choose Easy-A ,‡,† and closing of tubes, which exposes the lab to contamination High-Fidelity PCR Master Mix . This 2x formulation contains the risk. The Easy-A high-fidelity
Load more

Recommended publications
  • RT-PCR Protocol Utilizing RT-PCR
    Protocol TD-P Revision 1.0 Creation Date: 2/18/2019 Revision Date: 4/24/2019 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Utilizing RT-PCR Kit Introduction Our RT-PCR Kit combines two powerful mixtures: i) 5x Master Mix and ii) Hot start Taq 2x master mix with a standard buffer for 2-step RT-PCR. The two mixtures require minimal handling during the reaction setup and offer consistent and robust RT-PCR reactions. First strand cDNA synthesis is achieved by using the 5x Master Mix, which contains Reverse Transcriptase, recombinant RNase inhibitor, dNTPs, an optimized buffer, MgCl2 and protein stabilizers. Reverse Transcriptase is a recombinant MMLV reverse transcriptase with reduced RNase H activity and increased thermostability. This kit also provides two optimized primers and nuclease-free water. An anchored Oligo-dT primer [d(T)23VN] forces the primer to anneal to the beginning of the polyA tail and the random hexamer primer mix provides random and consistent priming sites covering the entire RNA templates including both mRNAs and non- polyadenylated RNAs. In addition, this kit is highly efficient at producing full-length cDNA from long RNA templates at temperatures between 42-55°C. The amplification step features a high quality Hot Start Taq DNA Polymerase, which offers higher fidelity and better amplification in a master mix format. RT-PCR product can be generated up to 6 kb. Our RT-PCR Kit can be used for cDNA synthesis followed by gene expression data validation. The advantages of our RT-PCR Kit include robust and active cDNA synthesis at temperatures up to 55°C and high efficiency at producing full-length cDNA from as little as 50 pg of total RNA.
    [Show full text]
  • Pfu DNA Polymerase Deoxynucleoside-Triphosphate: DNA Deoxynucleotidyl-Transferase (DNA-Directed); EC 2.7.7.7
    Version 060918 G:\products\productflyer\pcr\polymerasen\proof\descr_m3004_pfu_en.docx ENAXXON fon: +49(0)731 – 3608 123 X G fax: +49(0)731 – 3608 962 b i o s c i e n c e E-Mail: [email protected] Pfu DNA Polymerase Deoxynucleoside-triphosphate: DNA deoxynucleotidyl-transferase (DNA-directed); EC 2.7.7.7 Product Cat# Package size Pfu DNA Polymerase (proof-reading polymerase) M3004.0250 250 units Pfu DNA Polymerase (proof-reading polymerase) M3004.0500 2 x 250 units Pfu DNA Polymerase (proof-reading polymerase) M3004.1250 5 x 250 units Description The Genaxxon bioscience Pfu DNA Polymerase is a thermostable enzyme possessing 5‘-3‘ DNA polymerase and 3‘-5‘ proof reading exonuclease activities. It is isolated from the hyperthermophilic marine archae Pyroccocus furiosus (Pfu). The enzyme provides extremely high fidelity. Whereas the enzyme is not able to amplify long fragments as efficiently as the Genaxxon Taq polymerase because of its very high exonuclease activity. To overcome this restriction we recommend to use the Genaxxon ReproFast DNA Polymerase, which will provide a more robust synthesis of longer amplification products (Barnes et al. (1994) Proc. Natl. Acad. Sci., USA 91, 2216-2220). Use of the Genaxxon Pfu DNA Polymerase in amplification results in blunt-ended products, which is not recommended for cloning into T/A vectors. Concentration: 2.5 units/µL Unit definition: One unit is defined as the amount of enzyme that incorporates 10nmoles of dNTPs into acid-insoluble form in 30 min at 72°C under the assay conditions (25mM TAPS (tris-(hydroxymethyl)methyl-amino-propane-sulphonic acid, sodium salt) pH9.3 (at 25°C), 50mM KCl, 2mM MgCl2, 1mM ß-mercaptoethanol) and activated calf thymus DNA as substrate.
    [Show full text]
  • Biotechnology Explorer™
    Biotechnology Explorer™ GAPDH PCR Module Instruction Manual Catalog #166-5010EDU explorer.bio-rad.com This kit is shipped at 4°C. Open immediately upon arrival and store reagents at –20°C within 2 weeks. Duplication of any part of this document permitted for classroom use only. Please visit explorer.bio-rad.com to access our selection of language translations for Biotechnology Explorer kit curricula. For technical support, call your local Bio-Rad office or, in the U.S., call 1-800-424-6723 Dear Educator: Amplification as the path to visualization of DNA Molecular biologists are faced with the classic needle in a haystack problem. Often we must find a few copies of a piece of DNA that code for a given gene in the haystack of DNA comprising the entire genome. Even if there are a thousand copies of the same piece of DNA it is often still difficult to locate them. However by selectively amplifying only that specific piece of DNA we can separate it from the rest of the DNA and visualize it. Once we can visualize the DNA, we can use the tools of molecular biology to open the whole vista of genetic engineering. We can work with the DNA to make discoveries in science, agriculture, and medicine. The method used to amplify the DNA is the polymerase chain reaction (PCR). PCR is capable of repeatedly doubling the amount of specific DNA. After many PCR cycles a million-fold or billion-fold times as much DNA is generated. Because of the increasing use of PCR in science it is important to provide students with an understanding of the basic principles and applications of PCR.
    [Show full text]
  • The Heterodimeric Primase from the Euryarchaea Pyrococcus Abyssi: A
    Journal of Molecular Biology Archimer, archive institutionnelle de l’Ifremer Article in Press http://www.ifremer.fr/docelec/ Acceptation date : 5 October 2007 http://dx.doi.org/10.1016/j.jmb.2007.10.015 © 2007 Elsevier ailable on the publisher Web site The heterodimeric primase from the Euryarchaea Pyrococcus abyssi: a multifunctional enzyme for initiation and repair ? Magali Le Breton1, Ghislaine Henneke1, Cédric Norais2, Didier Flament1, Hannu Myllykallio2, Joël Querellou1 and Jean-Paul Raffin1,∗. 1. Ifremer, UMR6197, Laboratoire de Microbiologie des Environnements Extrêmes, BP 70, F-29280 blisher-authenticated version is av Plouzané, France. 2. Institut de Génétique et Microbiologie, UMR8621, Université Paris-sud, 91405 Orsay, France ∗Corresponding author: Jean Paul Raffin, Ifremer, Laboratoire de Microbiologie des Environnements Extrêmes, BP 70, 29280 Plouzané, France. Phone: 33(0)2 98 22 45 39 ; [email protected] Abstract: We report the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of two proteins, Pabp41 and Pabp46, showing sequence similarities respectively to the p49 and p58 subunits of the eukaryotic polymerase α-primase complex. Both subunits were expressed, purified and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribo- and ccepted for publication following peer review. The definitive pu deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+.
    [Show full text]
  • High-Fidelity Amplification Using a Thermostable DNA Polymerase Isolated from P’~Ococcusfuriosus
    Gette, 108 (1991) 1-6 8 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/SO3.50 GENE 06172 High-fidelity amplification using a thermostable DNA polymerase isolated from P’~OCOCCUSfuriosus (Polymerase chain reaction; mutation frequency; lack; proofreading; 3’40-5 exonuclease; recombinant DNA; archaebacteria) Kelly S. Lundberg”, Dan D. Shoemaker*, Michael W.W. Adamsb, Jay M. Short”, Joseph A. Serge* and Eric J. Mathur” “ Division of Research and Development, Stratagene, Inc., La Jolla, CA 92037 (U.S.A.), and h Centerfor Metalloen~~vme Studies, University of Georgia, Athens, GA 30602 (U.S.A.) Tel. (404/542-2060 Received by M. Salas: 16 May 1991 Revised/Accepted: 11 August/l3 August 1991 Received at publishers: 17 September 1991 -.-- SUMMARY A thermostable DNA polymerase which possesses an associated 3’-to-5’ exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10s.~ of the number of mutations obtained from similar amplifications performed with Tuq DNA polymerase. The PCR fidelity assay is based on the ampli~cation and cloning of lad, lac0 and IacZor gene sequences (IcrcIOZa) using either ffil or 7ii~irqDNA poIymerase. Certain mutations within the lucf gene inactivate the Lac repressor protein and permit the expression of /?Gal. When plated on a chromogenic substrate, these Lacl - mutants exhibit a blue-plaque phenotype.
    [Show full text]
  • Pfu DNA Polymerase
    344PR-01 G-Biosciences, St Louis, MO. USA ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected] A Geno Technology, Inc. (USA) brand name Pfu DNA Polymerase INTRODUCTION Pfu DNA polymerase, derived from the hyperthermophilic archae Pyrococcus furiosus, has superior thermostability and proofreading properties compared to other thermostable polymerase. Its molecular weight is 90 kD. It can amplify DNA target up to 2kb. The elongation velocity is 0.2~0.4kb/min (70~75°C). Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. Using Pfu DNA polymerase in your PCR reactions results in blunt-ended PCR products, which are ideal for cloning into blunt-ended vectors. Pfu DNA polymerase is superior for techniques that require high-fidelity DNA synthesis. ITEM(S) SUPPLIED Cat. # 786-816 Pfu DNA Polymerase (2.5U/µl) 500U 10X Pfu Buffer (Mg2+ plus) 2 x 1.4ml 6X Loading Buffer 1ml STORAGE CONDITIONS The kit is shipped at ambient temperature. Upon arrival, store at -20oC. Storage buffer is 20mM Tris.HCl (pH8.0), 100mM KCl, 3mM MgCl2, 1mM DTT, 0.1% Nonidet® P-40, 0.1% Tween® 20, 0.2mg/ml BSA, 50% (v/v) glycerol. 10X Pfu BUFFER 200mM Tris-HCl (pH8.8), 100mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton® X-100 and 1mg/ml BSA. UNIT DEFINITION One unit (U) of Pfu polymerase is defined as the amount of enzyme needed to catalyze the incorporation of 10 nanomoles of deoxyribonucleotides into acid-insoluble material in 30 minutes at 70°C using herring sperm DNA as a substrate.
    [Show full text]
  • High-Level Expression, Purification, and Thermus Aquatlcus DNA
    Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquatlcus DNA Polymerase and a Truncated Form Deficient in 5' to 3' Exonuclease Activity Frances C. Lawyer, 1 Susanne Stoffel, 1 Randall K. Saiki, 2 Sheng-Yung Chang, 3 Phoebe A. Landre, ~ Richard D. Abrarnson, 1 and David H. Gelfand 1 1Program in Core Research and Departments of 2Human Genetics and 3Infectious Disease, Roche Molecular Systems, Alameda, California 94501 The Thermus aquaticus DNA poly- life of 9 min at 97.5~ The Stoffel I in the native host is quite low (0.01- merase I (Taq Pol I) gene was cloned fragment has a half-life of 21 min at 0.02% of total protein). The cloning and into a plasmid expression vector that 97.5~ Taq Pol I contains a polymer- expression of full-length 94-kD Taq Pol I utilizes the strong bacteriophage ization-dependent 5' to 3' exonu- in E. coli under control of the E. coli lac PL promoter. A truncated form of Taq clease activity whereas the Stoffel promoter r or the tac promoter (7~ has Pol I was also constructed. The two fragment, deleted for the 5' to 3' ex- been reported. Because polymerase constructs made it possible to com- onuclease domain, does not possess yields in these constructs were low pare the full-length 832-amino-acid that activity. A comparison is made (-0.01% of total protein in our initial Taq Pol I and a deletion derivative among thermostable DNA poly- construct; see ref.
    [Show full text]
  • BIOTOOLS Pfu DNA POLYMERASE (1 U/Μl)
    WARRANTY Products are guaranteed to conform to the quality and content indicated on each vial and external labels during their shelf life. BIOTOOLS obligation and purchaser’s rights under this warranty are limited to the replacement by BIOTOOLS of any product that is shown defective in fabrication, and that must be returned to BIOTOOLS, freight prepaid, or at BIOTOOLS’ option, replacement of the purchasing price. Any complaint on damaged goods during transport must be directed to the handling or transport agent. Product for Research Use Only. This product must be used by qualified professionals only. It is the responsibility of the user to ascertain that a given product is adequate for a given application. Any product not fulfilling the specifications included in the product sheet will be replaced. This warranty limits our responsibility to the replacement of the product. No other warranties, of any kind, express or implied, including, without limitation, implicit warranties of commercialisation ability or adequacy for a given BIOTOOLS Pfu DNA POLYMERASE (1 U/l) purpose, are provided by BIOTOOLS. BIOTOOLS will not be held responsible for any direct, indirect, consequential or incidental damage resulting of the use, misuses, results of the use or inability to use any product. Produced by: REF. FORMAT CONTENT BIOTOOLS, Biotechnological & Medical Laboratories, S.A. have been evaluated and certified to accomplish ISO 9001:2000 requirements for the following activities: Research and development of biotechnology products and manufacture of biotechnology and in vitro products. Valle de Tobalina – 52 – Nave 39, 28021 Madrid – Spain BIOTOOLS Pfu DNA Polymerase (1 U/µl) 10.501 100 U © 2008 BIOTOOLS, Biotechnological & Medical Laboratories, S.A.
    [Show full text]
  • The Human TTAGGG Repeat Factors 1 and 2 Bind to a Subset of Interstitial Telomeric Sequences and Satellite Repeats
    npg Extratelomeric binding of TRF1 and TRF2 Cell Research (2011) 21:1028-1038. 1028 © 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats Thomas Simonet1, Laure-Emmanuelle Zaragosi2, Claude Philippe3, Kevin Lebrigand2, Clémentine Schouteden1, Adeline Augereau1, 3, Serge Bauwens1, Jing Ye1, 3, Marco Santagostino4, Elena Giulotto4, Frederique Magdinier1, Béatrice Horard1, Pascal Barbry2, Rainer Waldmann2, Eric Gilson1, 3, 5 1Laboratoire de Biologie Moléculaire de la Cellule-UMR 5239 CNRS/ENS Lyon/ Université Lyon, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, Lyon 69364, France; 2CNRS and University of Nice Sophia Antipolis, Institut de Pharmacologie Molé- culaire et Cellulaire, 06560 Sophia Antipolis, France; 3Laboratory of Biology and Pathology of Genomes of University of Nice Sophia-Antipolis, CNRS UMR6267/INSERM U998, Faculty of Medicine, Nice, France; 4Dipartimento di Genetica e Microbiologia Adriano Buzzati-Traverso, Università di Pavia, Pavia, Italy; 5Department of Medical Genetics, CHU of Nice, Nice, France The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mech- anisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian te- lomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind.
    [Show full text]
  • Cloning the Pfu DNA Polymerase from DNA Contaminants in Preparations of Commercial Pfu DNA Polymerase
    African Journal of Microbiology Research Vol. 7(9), pp. 745-750, 26 February, 2013 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.1124 ISSN 1996 0808 ©2013 Academic Journals Full Length Research Paper Cloning the Pfu DNA polymerase from DNA contaminants in preparations of commercial Pfu DNA polymerase Ji Hye Heo and Suhng Wook Kim* Department of Biomedical Science, College of Health Sciences, Korea University, Seoul 136-703, Korea. Accepted 25 February, 2013 The Pfu DNA polymerase gene was cloned from commercial Pfu DNA polymerase by semi-nested polymerase chain reaction (PCR) using a contaminant template in the commercial Pfu DNA polymerase and the activity of the Pfu DNA polymerase itself. We confirmed the occurrence of residual bacterial DNA in standard Pfu DNA polymerase preparations and demonstrated that the commercial enzyme can be used directly as a PCR template for cloning, thus eliminating the laborious and time-consuming steps of cell recovery and DNA extraction. A simple method was developed for the purification of Pfu DNA polymerase by the heat-mediated (100°C, 10 min) lysis of Escherichia coli and denaturation of E. coli proteins, exploiting using the extremely thermostable properties of Pfu DNA polymerase, followed by chromatography on heparin columns. The whole purification process could be achieved within 2 h using one type of buffer, with no need for sonication or the use of toxic reagents. This advantageous alternative method is convenient and faster than previously reported methods. Key words: Pfu DNA polymerase, DNA contamination, cloning, purification. INTRODUCTION Pfu DNA polymerase is an archaeal family B or human α- (Lundberg et al., 1991).
    [Show full text]
  • Characterisation of the ATP-Dependent Phosphofructokinase Gene Family from Arabidopsis Thaliana
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 581 (2007) 2401–2410 Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana Angelika Mustropha,1, Uwe Sonnewaldb, Sophia Biemeltb,* a Humboldt-University Berlin, Institute of Biology, AG Plant Physiology, Philippstrasse 13, 10115 Berlin, Germany b Friedrich-Alexander University Erlangen-Nuremberg, Department of Biochemistry, Staudtstrasse 5, 91058 Erlangen, Germany Received 1 March 2007; revised 15 April 2007; accepted 16 April 2007 Available online 30 April 2007 Edited by Mark Stitt addition to the widely distributed ATP-dependent phospho- Abstract Plants possess two different types of phosphofructo- kinases, an ATP-dependent (PFK) and a pyrophosphate-depen- fructokinase (PFK, EC 2.7.1.11), in plants and a number of dent form (PFP). While plant PFPs have been investigated in prokaryotes a second type of phosphofructokinase is present detail, cDNA clones coding for PFK have not been identified using pyrophosphate (PPi) instead of ATP as phosphoryl in Arabidopsis thaliana. Searching the A. thaliana genome re- donor (pyrophosphate-fructose-6-phosphate-phosphotransfer- vealed 11 putative members of a phosphofructokinase gene fam- ase, PFP, EC 2.7.1.90). Phosphorylation of Fru6P catalyzed by ily. Among those, four sequences showed high homology to the PFK is virtually irreversible in vivo, while PFP reacts near alpha- or beta-subunits of plant PFPs. Seven cDNAs resulted equilibrium and catalyzes the reaction in both directions. In in elevated PFK, but not PFP activity after transient expression addition a third, ADP-dependent phosphofructokinase has in tobacco leaves suggesting that they encode Arabidopsis PFKs.
    [Show full text]
  • PCR Protocol with Pfu DNA Polymerase-Final
    Protocol TD-P Revision 1.0 Creation Date: 6/4/2018 Revision Date: 7/17/2018 Polymerase Chain Reaction (PCR) Utilizing Pfu DNA Polymerase Introduction Polymerase Chain Reaction (PCR) is a powerful technique used to amplify DNA through the use of the enzyme DNA Polymerase. GoldBio Pfu DNA Polymerase is a thermostable enzyme that has 5’→3’ DNA polymerase and 3’→5’ exonuclease (proofreading) activities. GoldBio Pfu DNA Polymerase retains the high fidelity, sensitivity and processivity with an error rate six-fold lower than Taq DNA polymerase. In addition, GoldBio Pfu DNA Polymerase exhibits significantly lower error rates than most other proofreading enzymes or DNA polymerase mixtures. GoldBio Pfu DNA Polymerase is supplied with a 10x PCR reaction buffer, containing MgCl 2, which produces a final Mg 2+ concentration of 1.5mM and 5x GC enhancer that enables efficient amplification of GC-rich templates up to 84%. GoldBio Pfu DNA Polymerase can be used in many applications including routine PCR cloning, primer extension, colony PCR, genotyping and amplification of high GC content DNA with the GC enhancer. Here, we describe a general protocol for the use of GoldBio Pfu DNA Polymerase. Materials GoldBio Pfu DNA Polymerase (GoldBio Catalog # P-665 or P-690 ) 10x PCR Buffer with Mg 2+ 5x GC enhancer 10mM dNTP (not supplied with P-665) Not supplied Primers H2O PCR Grade Note: The storage buffer contains 50mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% glycerol, pH 7.5 at 25°C. 2+ Note: 10x PCR Buffer with Mg contains 100mM Tris-HCl, pH 9.0, 15mM MgCl 2, 100mM KCl, 80mM (NH 4)2SO 4, 0.5% Igepal CA 630.
    [Show full text]