DOCSLIB.ORG

Cold Spring Harbor Laboratory Press - Molecular Cloning - Table of Contents

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cold Spring Harbor Laboratory Press - Molecular Cloning - Table of Contents Cold Spring Harbor Laboratory Press - Molecular Cloning - Table of Contents http://www.synthesisgene.com Table of Contents Chapter 1: Plasmids and Their Usefulness in Molecular Cloning Chapter 2: Bacteriophage and Its Vectors Chapter 3: Working with Bacteriophage M13 Vectors Chapter 4: Working with High-Capacity Vectors Chapter 5: Gel Electrophoresis of DNA and Pulsed-Field Agarose Chapter 6: Preparation and Analysis of Eukaryotic Genomic DNA Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells Chapter 8: In Vitro Amplification of DNA by the Polymerase Chain Reaction Chapter 9: Preparation of Radiolabeled DNA and RNA Probes Chapter 10: Working with Synthetic Oligonucleotide Probes Chapter 11: Preparation of cDNA Libraries and Gene Identification Chapter 12: DNA Sequencing Chapter 13: Mutagenesis Chapter 14: Screening Expression Libraries Chapter 15: Expression of Cloned Genes in Escherichia coli Chapter 16: Introducing Cloned Genes into Cultured Mammalian Cells Chapter 17: Analysis of Gene Expression in Cultured Mammalian Cells Chapter 18: Protein Interaction Technologies Protocols | Bioinformatics | Cautions | Trademarks | Forums Contact Us | Help | Logout | Members Home | My Account Buy The Book | Our Vision | Take The Tour | Newsletter | Search Copyright © 2001 by Cold Spring Harbor Laboratory Press. All rights reserved. No part of these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction modification, storage in a retrieval system or retransmission, in any form or by any means, electronic, mechanical, or otherwise, for reasons other than personal use, is strictly prohibited without prior written permission. http://www.molecularcloning.com/members/toc.jsp [2002-2-18 16:10:23] Cold Spring Harbor Laboratory Press - Molecular Cloning - Chapter 1 http://www.synthesisgene.com Chapter 1 Plasmids and Their Usefulness in Molecular Cloning Protocol 1: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS. Protocol 2: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterial cultures by treatment with alkali and SDS. Protocol 3: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS. Protocol 4: Preparation of Plasmid DNA by Small-scale Boiling Lysis Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X- 100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). Protocol 5: Preparation of Plasmid DNA by Large-scale Boiling Lysis Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X- 100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). Protocol 6: Preparation of Plasmid DNA: Toothpick Minipreparation Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. Protocol 7: Preparation of Plasmid DNA by Lysis with SDS Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS. Protocol 8: Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Crude preparations of plasmid DNA are first treated with lithium chloride and RNase (to remove RNA). The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2. Protocol 9: Purification of Plasmid DNA by Chromatography The following table summarizes the salient features of many of the commercial resins that are currently available for plasmid purification. Individual manufacturers supply detailed instructions, which should be followed to the letter. Protocol 10: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl- Ethidium Bromide Gradients: Continuous Gradients Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl. The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 µg/ml. During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities. Protocol 11: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl- Ethidium Bromide Gradients: Discontinuous Gradients A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl). During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities. Protocol 12: Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents Ethidium bromide is removed from DNA by phase extraction with organic solvents. Protocol 13: Removal of Ethidium Bromide from DNA by Ion-exchange Chromatography Ethidium bromide is removed from DNA by chromatography through a cation-exchange resin. Protocol 14: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sodium chloride. This method was devised by Brian Seed when he was a graduate student at Harvard University. Protocol 15: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Chromatography through Sephacryl S-1000 Contamination of plasmid DNA by small fragments of nucleic acid is reduced dramatically by size-exclusion chromatography through Sephacryl S-1000. Protocol 16: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. Protocol 17: Directional Cloning into Plasmid Vectors Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. Protocol 18: Attaching Adaptors to Protruding Termini Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. Protocol 19: Blunt-ended Cloning into Plasmid Vectors http://www.molecularcloning.com/members/chapter.jsp?chapter=112 (1 / 3) [2002-2-18 16:10:49] Cold Spring Harbor Laboratory Press - Molecular Cloning - Chapter 1 http://www.synthesisgene.com Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E. coli. The maximum number of "correct" clones can generally be obtained from ligation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 µg/ml. Blunt-end ligation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and polyamines such as spermidine. Protocol 20: Dephosphorylation of Plasmid DNA During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5´-phosphate residues from both termini of the linear, double- stranded plasmid DNA with alkaline phosphatase. Protocol 21: Addition of Synthetic Linkers to Blunt-ended DNA Linkers are small self-complementary pieces of synthetic DNA, usually 8-16 nucleotides in length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site. Linkers are used to equip blunt-ended termini of DNA with restriction sites as an aid to cloning. Protocol 22: Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. Protocol 23: The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation This procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/mg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. Protocol 24: The Inoue Method for Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells This protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/mg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a
Load more

Recommended publications
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • A Visual Demonstration of Plasmid DNA Preparation from Bacteria
    HOW-TO-'DO-IT A Visual Demonstration of Plasmid DNA Preparation from Bacteria * RALPH L. KEIL, LAURA K. PALMER Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 Unprecedented advances in unraveling mysteries of life have disease, or other areas the students or instructor want to develop. occurred as a result of the molecular biology revolution. Some Since many students know someone with diabetes (in many cases major achievements of this revolution include new methods for the they know a classmate who has diabetes or one of them may even production of therapeutic compounds, insights into mechanisms suffer from the disease), this example provides an opportunity to of gene function and regulation, and the complete sequencing of get students to actively participate in discussion, regardless of their the genomes of numerous organisms including humans. A critical academic abilities. foundation of this revolution is the ability to use the bacterium An alternative introduction is to discuss consequences of Escherichia coli (E. coli) as a factory to produce large amounts of sequencing the entire three billion base pairs of DNA of the virtually any DNA of interest by inserting it into a small, extrachro human genome (http://www.ornl.gov/sci/techresources/Human mosomal circle of DNA called a plasmid. The ease and reproducibil Genome/home.shtml). The current and potential uses of this ity of plasmid DNA isolation from bacteria permits numerous labs technology in forensic science, health care, and business decisions to conduct molecular biology experiments and greatly accelerates are all areas that may be discussed depending on what focus the progress in understanding complex biological processes.
    [Show full text]
  • Control of Ph During Plasmid Preparation by Alkaline Lysis of Escherichia Coli
    Analytical Biochemistry 378 (2008) 224–225 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Notes & Tips Control of pH during plasmid preparation by alkaline lysis of Escherichia coli Cheri Cloninger, Marilyn Felton 1, Bonnie Paul 1, Yasuko Hirakawa, Stan Metzenberg * Department of Biology, California State University, Northridge, Northridge, CA 91330, USA article info abstract Article history: Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ‘‘ghost bands” Received 9 March 2008 of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing Available online 11 April 2008 the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.0, we were able to stabilize the pH during lysis and obtain plasmid that is suitably pure for restriction digestion and DNA sequencing. Ó 2008 Elsevier Inc. All rights reserved. The extraction of plasmids from Escherichia coli is one of the most 2. To each 1 ml of cell suspension, 1 ml of a lysis solution consist- commonly used methods in molecular biology, and detergent lysis of ing of 1% (w/v) sodium dodecyl sulfate (SDS) and 0.5 M L-argi- cells in 0.1 M sodium hydroxide (NaOH,2 final) is the most wide- nine (pH 11.7) is added, and the tube is capped and rocked spread approach [1,2]. The alkalinity denatures the chromosomal briefly to mix. The lysate is allowed to sit undisturbed for 5 min.
    [Show full text]
  • Molecular Biology Services Price List Denmark; Valid from 01.09.2017
    Molecular Biology Services Price List Denmark; valid from 01.09.2017 Gene Synthesis Service Delivery times for Gene Synthesis Service: Standard Genes up to 1000 bp: 6 business days; guaranteed: 12 business days Standard Genes ≤ 3000 bp: 15 - 20 business days Standard Genes > 3000 bp: 4 additional business days/kb; Subcloning: additional 5 - 10 business days Express Genes up to 1000 bp: 4 business days Express Genes up to 1500 bp: 6 business days; up to 2000 bp: 7 days; Express Genes up to 3000 bp: 11 business days; up to 4000 bp: 13 days; Express subcloning: 4 business days Delivery time for complex gene varies. Dependent on the complexity of the gene, usually it takes 5-10 business days longer than estimated for standard genes. Part ID Service Description Price [DKK] 5001-000010 Short Standard Genes (≤ 500 bp) 1,125.00 / gene 5001-000016 Standard Genes (501-1000 bp) 2.40 / base pair 5001-000012 Long Standard Genes (1001-3000bp) 2.40 / base pair 5001-000019 Long Standard Genes (3001-4000bp) 2.70 / base pair 5001-000119 Long Standard Gene (4001-5000bp) 3.00 / base pair 5001-000219 Long Standard Gene (5001-6000bp) 3.15 / base pair 5001-000319 Long Standard Gene (6001-7000bp) 3.53 / base pair 5001-000419 Long Standard Gene (7001-8000bp) 3.53 / base pair 5001-000519 Long Standard Gene (8001-9000bp) 3.53 / base pair 5001-000619 Long Standard Gene (9001-10000bp) 3.53 / base pair 5001-000719 Long Standard Gene > 10 kb on request 5001-000021 Complex Genes on request 5001-000230 Express Fee Short Standard Gene (≤ 500 bp) 3,000.00 / gene 5001-000232
    [Show full text]
  • Genomic/Plasmid Dna and Rna)
    NPTEL – Bio Technology – Genetic Engineering & Applications MODULE 4- LECTURE 1 ISOLATION AND PURIFICATION OF NUCLEIC ACIDS (GENOMIC/PLASMID DNA AND RNA) 4-1.1. Introduction Every gene manipulation procedure requires genetic material like DNA and RNA. Nucleic acids occur naturally in association with proteins and lipoprotein organelles. The dissociation of a nucleoprotein into nucleic acid and protein moieties and their subsequent separation, are the essential steps in the isolation of all species of nucleic acids. Isolation of nucleic acids is followed by quantitation of nucleic acids generally done by either spectrophotometric or by using fluorescent dyes to determine the average concentrations and purity of DNA or RNA present in a mixture. Isolating the genetic material (DNA) from cells (bacterial, viral, plant or animal) involves three basic steps- • Rupturing of cell membrane to release the cellular components and DNA • Separation of the nucleic acids from other cellular components • Purification of nucleic acids 4-1.2. Isolation and Purification of Genomic DNA Genomic DNA is found in the nucleus of all living cells with the structure of double- stranded DNA remaining unchanged (helical ribbon). The isolation of genomic DNA differs in animals and plant cells. DNA isolation from plant cells is difficult due to the presence of cell wall, as compared to animal cells. The amount and purity of extracted DNA depends on the nature of the cell. The method of isolation of genomic DNA from a bacterium comprises following steps (Figure 4-1.2.)- 1. Bacterial culture growth and harvest. 2. Cell wall rupture and cell extract preparation. Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 57 NPTEL – Bio Technology – Genetic Engineering & Applications 3.
    [Show full text]
  • An Improved System for Competent Cell Preparation and High Efficiency Plasmid Transformation Using Different Escherichia Coli Strains
    Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.8 No.1, Issue of April 15, 2005 © 2005 by Pontificia Universidad Católica de Valparaíso -- Chile Received June 8, 2004 / Accepted January 13, 2005 TECHNICAL NOTE An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains Zhiming Tu China-UK HUST-Res Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 27 87548885 E-mail: [email protected] Guangyuan He* China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 85 27 87548885 E-mail: [email protected] Kexiu X. Li China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 27 87548885 Mingjie J. Chen China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 27 87548885 Junli Chang China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 2787548885 Ling Chen China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 2787548885 Qing Yao China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 2787548885 Dongping P.
    [Show full text]
  • SGI-DNA-Bioxp-Lucigen-Webinar
    Accelerate your Research with Synthetic DNA and Hands-free Cloning Brought to you in collaboration with Lucigen and SGI-DNA Introduction Julie K. Robinson Synthetic Genomics, SGI-DNA Sr. Product Manager, Synthetic Biology The Potential of Synthetic Biology C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 2 Synthetic Genomics Collaborative Efforts Advance C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 3 Advancing Genomic Research Solutions, SGI-DNA Reagents Instruments ● Gibson Assembly® enables rapid assembly ● BioXp™ 3200 – an automated, benchtop of multiple DNA fragments genomics workstation ● Vmax electrocompetent cells for protein ● Create genes, genetic elements and complex expression genetic sequences ● Uses electronically transmitted sequence to build genes Services Bioinformatics ● Synthesize custom DNA of varying size ● Expert bioinformatics support, which and complexity includes whole genome annotation, gene expression analysis, and other types of ● Utilizes patented Gibson Assembly® sequence design method and error correction technology ● Archetype® software enables ‘omics-based ● Cell engineering services understanding of data to enable researchers ● NGS and plasmid preparation services to discover, analyze and build genes C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 4 Common Genomic applications Applications of DNA Cloning Study of Genomes & Transgenic Gene Expression Gene Therapy Organisms Biopharmaceutical Recombinant Protein Cell Engineering Research Production C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C.
    [Show full text]
  • Bacterial Culture & Plasmid Purification
    Genetic Tools and Reagents Bacterial Culture Media, Agar, Yeast Extract, Casein Peptone Plasmid Purification Bacterial Culture & Plasmid Purification Product Manual Bacterial Culture Media Protocols & Principle Plasmid Purification Protocols & Principle Bacterial Culture Media & Omni-Pure™ Mini-Prep Plasmid DNA Purification System For research use only. Not for use in diagnostic procedures for clinical purposes. Ordering Information Bacterial Culture Media Product Catalog No. Unit Size Agar Type A Bacterial Culture Grade, 100 g 40-3301-10 100 g Agar Type A Bacterial Culture Grade, 500 g 40-3301-05 100 g Agar Type A Bacterial Culture Grade, 1 kg 40-3301-01 1 kg Yeast Extract Bacterial Culture Grade, 100 g 40-4331-10 100 g Yeast Extract Bacterial Culture Grade, 500 g 40-4331-10 500 g Yeast Extract Bacterial Culture Grade, 1 k g 40-4331-10 1 kg Casein Peptone (Type 1) Bacterial Culture Grade, 100 g 40-3305-10 100 g Casein Peptone (Type 1) Bacterial Culture Grade, 500 g 40-3305-05 500 g Casein Peptone (Type 1) Bacterial Culture Grade, 1 kg 40-3305-01 1 kg Omni-Pure Plasmid DNA Purification Systems; Mini-Prep Product Catalog No. Unit Size* Omni-Pure Plasmid DNA Purification System; Mini-Prep 40-4020-01 100 Omni-Pure Plasmid DNA Purification System; Mini-Prep 40-4020-05 500 *Mini-prep plasmid purification. M_Bacterial_Culture Page 2 of 20 Bacterial Culture Media & Omni-Pure™ Mini-Prep Plasmid DNA Purification System For research use only. Not for use in diagnostic procedures for clinical purposes. Bacterial Culture & Plasmid Purification Protocols
    [Show full text]
  • Bacterial Transformation
    TECHNIQUES IN MOLECULAR BIOLOGY – BACTERIAL TRANSFORMATION The uptake of exogenous DNA by cells that alters the phenotype or genetic trait of a cell is called transformation. For cells to uptake exogenous DNA they must first be made permeable so the DNA can enter the cells. This state is referred to as competency. In nature, some bacteria become competent due to environmental stresses. We can purposely cause cells to become competent by treatment with chloride salts of metal cations such as calcium, rubidium or magnesium and cold treatment. These changes affect the structure and permeability of the cell wall and membrane so that DNA can pass through. However, this renders the cells very fragile and they must be treated carefully while in this state. Competent cells vary in how well they take up DNA. We can express this The amount of cells transformed per 1 µg of DNA is called the transformation efficiency. Too little DNA can result in low transformation efficiencies, but too much DNA also inhibits the transformation process. Transformation efficiencies generally range from 1 x 104 to 1 x 107 transformed cells per µg of added DNA. E. coli bacteria are normally poisoned by the antibiotic ampicillin. Ampicillin inhibits synthesis of the bacterial cell wall (in bacteria like E. coli , found between the inner and outer cell membranes), resulting in bacteria that are very structurally weak. In the hypotonic media in which these cells grow, cells exposed to ampicillin will swell and burst or not grow at all. For cells to survive, they must include a means to break down the ampicillin.
    [Show full text]
  • QIAGEN® Plasmid Purification Handbook
    February 2021 QIAGEN® Plasmid Purification Handbook QIAGEN Plasmid Mini, Midi, Maxi, Mega, and Giga Kits For purification of ultrapure, transfection-grade plasmid DNA Sample to Insight__ Contents Kit Contents ............................................................................................................... 3 Shipping and Storage ................................................................................................. 4 Intended Use .............................................................................................................. 4 Safety Information ....................................................................................................... 5 Quality Control ........................................................................................................... 5 Introduction ................................................................................................................ 6 Principle and procedure .................................................................................... 6 Equipment and Reagents to Be Supplied by User ............................................................ 8 Important Notes .......................................................................................................... 9 Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit .............. 17 Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits .............................................................................................................................
    [Show full text]
  • Molecular Cloning
    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.
    [Show full text]