Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual

Size: px
Start display at page:

Download "Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual"

Transcription

1 Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual I. Purpose...1 II. Introduction...1 III. Inhibition of Bacterial Growth Protocol...2 IV. Inhibition of in vitro Translation Protocol...4 V. References...6 This student manual is! available online only. Reproduction for noncommercial educational purposes only. Copyright 2007 Promega Corporation. All rights reserved. I. Purpose Many common antibiotics inhibit bacterial growth by inhibiting protein synthesis. The purpose of this experiment is to demonstrate the effects of various compounds on bacterial growth and translation in an in vitro prokaryotic protein expression system. II. Introduction The central dogma of molecular biology states that information is relayed from DNA to RNA to protein in the cell. During transcription, RNA polymerase makes an RNA copy of the DNA template. Translation is the process by which the ribosome uses the resulting RNA molecule as a template to synthesize a protein. This protocol describes experiments to explore the effects of antibiotics on bacterial growth and protein synthesis. Bacterial growth in the presence of antibiotics will be monitored by streaking Escherichia coli cells onto growth medium in a Petri dish and placing several disks containing antibiotics onto the medium. In areas of the dish where bacterial growth is not inhibited, a lawn of bacteria will grow, and the growth medium will appear opaque. Areas where growth is inhibited will appear translucent; the translucent area is known as the zone of inhibition. The effect of antibiotics on protein synthesis will be investigated using an E. coli S30 extract capable of in vitro transcription and translation. S30 extracts are prepared from E. coli and contain the necessary proteins for both transcription and translation (1,2). To initiate the reaction, a DNA template and other components necessary for transcription and translation are added to the S30 extract. The DNA template is transcribed as RNA, and the RNA is translated to produce protein. In these experiments, the DNA template is the pbestluc Vector, which contains the eukaryotic firefly luciferase gene (the luc gene) positioned downstream from the tac promoter and a ribosome binding site (Figure 1). Luciferase is the enzyme that produces light in luminous beetles, including fireflies. The light produced by firefly luciferase is easily measured using a luminometer, an instrument that measures light output. The amount of light produced in the enzymatic reaction is proportional to the amount firefly luciferase protein synthesized. Thus, light output is an indication of the level of protein synthesis. In this laboratory exercise we will examine the effects of various compounds on bacterial growth and protein synthesis in an in vitro transcription/translation system. 8/07 Page 1

2 Hind III 4396 Ptac luc Cla I 1374 pbestluc Vector (4486bp) Xho I 1717 Amp r ori 0693VB Figure 1. Vector circle map of the pbestluc Vector. III. Inhibition of Bacterial Growth Protocol Page 2 Materials Required two Mueller Hinton agar plates at room temperature E. coli culture with an absorbance in the range of at 600 nm antibiotic disks and a blank disk small beaker with alcohol and forceps sterile cotton swabs Bunsen burner metric ruler markers to label plates! Don t handle antibiotic disks if you are allergic to that form of antibiotic. Protocol Day 1 1. Inoculate one Mueller Hinton agar plate with E. coli. Dip a sterile cotton swab into the overnight culture of E. coli, and wipe off any excess on the inside of the tube. Inoculate a Mueller Hinton agar plate by streaking the entire surface of the plate with the swab, turning the plate 90, swabbing a second time, turning the plate 45 and swabbing a third time. Run the swab around the circumference of the plate. Be sure to cover the entire plate. Discard the swab. 2. Repeat Step 1 using a fresh sterile swab to inoculate the second Mueller Hinton agar plate. 3. Allow the plates to dry for 5 minutes before placing the antibiotic disks on the plate surface. 4. Apply four antibiotic disks to the agar surface of one plate using sterile forceps. To sterilize the forceps, dip them in alcohol, letting the excess drip into the beaker, and pass them through the Bunsen burner flame. Allow the alcohol to burn off. Be sure to space the disks at least 4 5 cm apart to prevent overlapping zones of growth inhibition. Press each disk gently with sterile forceps so that the disk makes good contact with the surface of the agar. Note: Each antibiotic disk will be marked with an abbreviation (Table 1). The blank disk has no markings. 8/07

3 5. Repeat Step 4 to apply the other two antibiotic disks and the blank disk to the second Mueller Hinton agar plate. 6. Press each disk gently with sterile forceps so that the disk makes good contact with the surface of the agar. 7. Cover the plates. Label the plates with your name, date and organism name. 8. Invert the plates, and incubate them at C for hours. Day 2 1. Remove the plates from the incubator. With a metric ruler, measure the zone of inhibition, the distance between the edge of the disk and edge of bacterial growth, at the widest point. Note: Results may be easier to read if plates are placed on a black background or a light box. 2. Record the results in Table 1. Record whether the compound inhibited bacterial growth. Results Table 1. Effect of Compounds on Bacterial Growth. Compound ampicillin chloramphenicol streptomycin tetracycline erythromycin neomycin blank Abbreviation AM C S TE E N Zone of Inhibition (mm) Inhibited Bacterial Growth (Yes or No?) Discussion 1. Which compounds were the most effective in inhibiting growth of E. coli, based on the width of the zone of inhibition? Which were the least effective? 2. Based on the mechanisms of action listed in Table 2, why was bacterial growth inhibited? Can you explain why some of the antibiotics tested were less effective than others at inhibiting growth of E coli? Would you have expected similar results if you had tested Staphylococcus aureus in your assay? Table 2. Modes of Action. Compound Ampicillin Chloramphenicol Streptomycin Tetracycline Erythromycin Neomycin Cycloheximide Mechanism of Action Inhibits cell wall synthesis by inhibiting formation of the peptidoglycan cross-link. Inhibits prokaryotic peptidyl transferase. Inhibits prokaryotic peptide chain initiation, and induces mrna misreading. Inhibits prokaryotic aminoacyl-trna binding to the ribosome small subunit. Inhibits prokaryotic peptide chain initiation. Inhibits prokaryotic translocation through the ribosome large subunit. Inhibits eukaryotic peptidyl transferase. 8/07 Page 3

4 IV. Inhibition of in vitro Transcription/Translation Protocol Materials Required E. coli S30 Extract System for Circular DNA, which contains: 175 µl Amino Acid Mixture Minus Cysteine, 1 mm 175 µl Amino Acid Mixture Minus Methionine, 1 mm 450 µl S30 Extract, Circular (3 150 µl) 750 µl S30 Premix Without Amino Acids 10 µl pbestluc DNA, Circular (1 µg/µl) pbestluc DNA, Circular (0.5 µg/µl) Steady-Glo Luciferase Assay System 1.5 ml microcentrifuge tubes nuclease-free water room-temperature distilled water luminometer or multimode instrument capable of measuring luminescence in vitro Transcription/Translation Protocol Use a fresh pipet tip for each reagent addition. 1. Prepare enough master mix for 9 in vitro transcription/translation reactions by Volume Per Component Reaction pbestluc DNA (0.5 µg/µl) 1.0 µl Amino Acid Mixture Minus Cysteine, 1mM (mix gently prior to use) 2.5 µl Amino Acid Mixture Minus Methionine, 1mM (mix gently prior to use) 2.5 µl S30 Premix Without Amino Acids (mix gently prior to use) 20 µl S30 Extract, Circular 2 (mix gently prior to use) 15 µl Nuclease-free water 8.0 µl Final volume 50 µl combining the following reaction components. 2. Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the reaction mixture to the bottom of the tube. 3. Label eight 1.5 ml microcentrifuge tubes as reactions 1 to 8. Pipet 49 µl of the master mix prepared in Step 1 to each of the tubes. Number of Reactions 1 = Final Volume 1You will assemble 8 reactions in this exercise but will prepare enough master mix for 9 reactions. This should ensure that you have enough master mix. 2The extract can be viscous, so take care to pipet near the top of the liquid level and do not submerge the pipet tip to the bottom of the tube, as small droplets of liquid can adhere to the pipet tip. Page 4 8/07

5 4. Pipet 1.0 µl of the compound to a tube containing the master mix as follows: Compound Tube Number ampicillin 1 chloramphenicol 2 streptomycin 3 tetracycline 4 erythromycin 5 neomycin 6 cycloheximide 7 5. Add 1.0 µl of nuclease-free water to tube 8. Note: Tube 8 does not contain a compound and will act as the positive control. This reaction represents the maximum level of protein synthesis. 6. Vortex each reaction gently to mix. Incubate the reaction at 37 C for 60 minutes. 7. Stop the reaction by placing the tubes on ice for 5 minutes. Detection of Luciferase Protein 1. Label nine microcentrifuge tubes 1 to 9. Add 95 µl of distilled water to tubes Add 100 µl of distilled water to tube Add 5.0 µl of each translation reaction to tubes Add 100 µl of Steady-Glo Reagent that has been equilibrated to room temperature to each tube, and mix by pipetting. Note: Light intensity is a measure of the amount of luciferase present but also the enzymatic rate, which depends upon temperature. Be sure that the Steady-Glo Reagent has been equilibrated to room temperature (20 25 C) for reproducible luciferase assay readings. 5. Place the reaction in a luminometer or multimode instrument capable of measuring luminescence. Measure the luminescence and record the results in Table 3. Consult the appropriate operator's manual for operation of the luminometer, if necessary. Note: Luminescence is measured in terms of relative light units (RLU). Table 4. Effect of Test Compounds on in vitro Transcription/Translation. Tube Number Compound Raw Luminescence (RLU) Net Luminescence (RLU) Percent Inhibition of Protein Synthesis 1 ampicillin 2 chloramphenicol 3 streptomycin 4 tetracycline 5 erythromycin 6 neomycin 7 cycloheximide 8 no compound 9 no compound; no translation reaction 8/07 Page 5

6 IV. Inhibition of in vitro Transcription/Translation Protocol (continued) 6. Calculate the net luminescence by subtracting the background luminescence measured in tube 9 from the experimental luminescence in tubes 1 8. Record net luminescence values in Table Calculate the percent of inhibition for each of the compounds using the equation given below. Reaction 8 did not contain an inhibitor, and the light output from this reaction represents the maximum level of protein synthesis. Net Luminescence in the Presence of Compound 100% Net Luminescence in the Absence of Compound Discussion 1. Which compounds were most effective at inhibiting protein synthesis in the E. coli S30 Extract System? 2. Did those compounds that inhibited in vitro transcription/translation also inhibit bacterial growth? Why? 3. Which compounds did not inhibit in vitro transcription and translation? Based on the mechanisms of action listed in Table 2, why was protein synthesis inhibited or not inhibited in each reaction? 4. What effect would you expect if you had placed a disk impregnated with cycloheximide on the Mueller Hinton agar plate in the growth inhibition study? Why? 5. The S30 Extract contains components required for transcription, including an RNA polymerase and NTPs. What is the role of each of these components in transcription? What is the role of the pbestluc DNA? 6. The S30 Premix Without Amino Acids contains components required for translation, including NTPs, trnas and an ATP-regenerating system. What is the role of each of these components in translation? V. References 1. Zubay, G. (1973) In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, Zubay, G. (1980) The isolation and properties of CAP, the catabolite gene activator. Meth. Enzymol. 65, Page 6 8/07

Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Instructor s Manual

Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Instructor s Manual Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Instructor s Manual I. Purpose and Concepts Covered...1 II. Inhibition of Bacterial Growth...2 A. Preparation for the Laboratory...2 B.

More information

Biological Sciences Initiative

Biological Sciences Initiative Biological Sciences Initiative HHMI Student Activities Measuring Antibiotic Resistance Introduction: You might be aware that antibiotics were once thought of as a magic bullet; a nearly perfect drug for

More information

GROWING BACTERIA INTRODUCTION

GROWING BACTERIA INTRODUCTION GROWING BACTERIA INTRODUCTION E. coli is a normal part of the bacterial flora of the human gut. It is not generally considered pathogenic, although some strains are highly toxic (recent food poisonings

More information

Disc Diffusion Susceptibility Methods

Disc Diffusion Susceptibility Methods Disc Diffusion Susceptibility Methods Introduction When a filter paper disc impregnated with a chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place

More information

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency

More information

Bacterial Transformation with Green Fluorescent Protein. Table of Contents Fall 2012

Bacterial Transformation with Green Fluorescent Protein. Table of Contents Fall 2012 Bacterial Transformation with Green Fluorescent Protein pglo Version Table of Contents Bacterial Transformation Introduction..1 Laboratory Exercise...3 Important Laboratory Practices 3 Protocol...... 4

More information

TransformAid Bacterial Transformation Kit

TransformAid Bacterial Transformation Kit Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection

More information

Lecture 6. Regulation of Protein Synthesis at the Translational Level

Lecture 6. Regulation of Protein Synthesis at the Translational Level Regulation of Protein Synthesis (6.1) Lecture 6 Regulation of Protein Synthesis at the Translational Level Comparison of EF-Tu-GDP and EF-Tu-GTP conformations EF-Tu-GDP EF-Tu-GTP Next: Comparison of GDP

More information

Transformation of the bacterium E. coli. using a gene for Green Fluorescent Protein

Transformation of the bacterium E. coli. using a gene for Green Fluorescent Protein Transformation of the bacterium E. coli using a gene for Green Fluorescent Protein Background In molecular biology, transformation refers to a form of genetic exchange in which the genetic material carried

More information

HiPer RT-PCR Teaching Kit

HiPer RT-PCR Teaching Kit HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The

More information

Enzymes: Amylase Activity in Starch-degrading Soil Isolates

Enzymes: Amylase Activity in Starch-degrading Soil Isolates Enzymes: Amylase Activity in Starch-degrading Soil Isolates Introduction This week you will continue our theme of industrial microbiologist by characterizing the enzyme activity we selected for (starch

More information

GENETIC TRANSFORMATION OF BACTERIA WITH THE GENE FOR GREEN FLUORESCENT PROTEIN (GFP)

GENETIC TRANSFORMATION OF BACTERIA WITH THE GENE FOR GREEN FLUORESCENT PROTEIN (GFP) GENETIC TRANSFORMATION OF BACTERIA WITH THE GENE FOR GREEN FLUORESCENT PROTEIN (GFP) LAB BAC3 Adapted from "Biotechnology Explorer pglo Bacterial Transformation Kit Instruction Manual". (Catalog No. 166-0003-EDU)

More information

Reverse Transcription System

Reverse Transcription System TECHNICAL BULLETIN Reverse Transcription System Instruc ons for use of Product A3500 Revised 1/14 TB099 Reverse Transcription System All technical literature is available on the Internet at: www.promega.com/protocols/

More information

BCH401G Lecture 39 Andres

BCH401G Lecture 39 Andres BCH401G Lecture 39 Andres Lecture Summary: Ribosome: Understand its role in translation and differences between translation in prokaryotes and eukaryotes. Translation: Understand the chemistry of this

More information

Transferring a Broth Culture to Fresh Broth

Transferring a Broth Culture to Fresh Broth Sterile Technique It is very important in microbiology to work with pure cultures. Unfortunately this is difficult. The world around us is covered with microorganisms. Microorganisms are even carried on

More information

Instructions. Torpedo sirna. Material. Important Guidelines. Specifications. Quality Control

Instructions. Torpedo sirna. Material. Important Guidelines. Specifications. Quality Control is a is a state of the art transfection reagent, specifically designed for the transfer of sirna and mirna into a variety of eukaryotic cell types. is a state of the art transfection reagent, specifically

More information

13.2 Ribosomes & Protein Synthesis

13.2 Ribosomes & Protein Synthesis 13.2 Ribosomes & Protein Synthesis Introduction: *A specific sequence of bases in DNA carries the directions for forming a polypeptide, a chain of amino acids (there are 20 different types of amino acid).

More information

From DNA to Protein. Proteins. Chapter 13. Prokaryotes and Eukaryotes. The Path From Genes to Proteins. All proteins consist of polypeptide chains

From DNA to Protein. Proteins. Chapter 13. Prokaryotes and Eukaryotes. The Path From Genes to Proteins. All proteins consist of polypeptide chains Proteins From DNA to Protein Chapter 13 All proteins consist of polypeptide chains A linear sequence of amino acids Each chain corresponds to the nucleotide base sequence of a gene The Path From Genes

More information

First Strand cdna Synthesis

First Strand cdna Synthesis 380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences

More information

Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein

Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein INTRODUCTION Green Fluorescent Protein (GFP) is a novel protein produced by the bioluminescent

More information

Specific problems. The genetic code. The genetic code. Adaptor molecules match amino acids to mrna codons

Specific problems. The genetic code. The genetic code. Adaptor molecules match amino acids to mrna codons Tutorial II Gene expression: mrna translation and protein synthesis Piergiorgio Percipalle, PhD Program Control of gene transcription and RNA processing mrna translation and protein synthesis KAROLINSKA

More information

Lecture 1 MODULE 3 GENE EXPRESSION AND REGULATION OF GENE EXPRESSION. Professor Bharat Patel Office: Science 2, 2.36 Email: b.patel@griffith.edu.

Lecture 1 MODULE 3 GENE EXPRESSION AND REGULATION OF GENE EXPRESSION. Professor Bharat Patel Office: Science 2, 2.36 Email: b.patel@griffith.edu. Lecture 1 MODULE 3 GENE EXPRESSION AND REGULATION OF GENE EXPRESSION Professor Bharat Patel Office: Science 2, 2.36 Email: b.patel@griffith.edu.au What is Gene Expression & Gene Regulation? 1. Gene Expression

More information

CLONING IN ESCHERICHIA COLI

CLONING IN ESCHERICHIA COLI CLONING IN ESCHERICHIA COLI Introduction: In this laboratory, you will carry out a simple cloning experiment in E. coli. Specifically, you will first create a recombinant DNA molecule by carrying out a

More information

DNA CAN BE TRANSFERRED BETWEEN BACTERIA GENETIC ENGINEERING USING RECOMBINANT DNA TECHNOLOGY

DNA CAN BE TRANSFERRED BETWEEN BACTERIA GENETIC ENGINEERING USING RECOMBINANT DNA TECHNOLOGY Bacterial Transformation DNA CAN BE TRANSFERRED BETWEEN BACTERIA Background Information Plasmid Transformed Cell Figure 1: Bacterial Transformation Quick Reference Abbreviations GFP pgfp gfp Green fl uorescent

More information

Module 3 Questions. 7. Chemotaxis is an example of signal transduction. Explain, with the use of diagrams.

Module 3 Questions. 7. Chemotaxis is an example of signal transduction. Explain, with the use of diagrams. Module 3 Questions Section 1. Essay and Short Answers. Use diagrams wherever possible 1. With the use of a diagram, provide an overview of the general regulation strategies available to a bacterial cell.

More information

ab185916 Hi-Fi cdna Synthesis Kit

ab185916 Hi-Fi cdna Synthesis Kit ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1

More information

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)

More information

Welcome to Implementing Inquirybased Microbial Project. Veronica Ardi, PhD

Welcome to Implementing Inquirybased Microbial Project. Veronica Ardi, PhD Welcome to Implementing Inquirybased Microbial Project Veronica Ardi, PhD Microbiology Laboratory Courses CourseSmart: ebook resources http://instructors.coursesmart.com/ Microbiology Laboratory Courses

More information

Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual

Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual F- 470S 20 cdna synthesis reactions (20 µl each) F- 470L 100 cdna synthesis reactions (20 µl each) Table of contents 1. Description...

More information

Induction of Enzyme Activity in Bacteria:The Lac Operon. Preparation for Laboratory: Web Tutorial - Lac Operon - submit questions

Induction of Enzyme Activity in Bacteria:The Lac Operon. Preparation for Laboratory: Web Tutorial - Lac Operon - submit questions Induction of Enzyme Activity in Bacteria:The Lac Operon Preparation for Laboratory: Web Tutorial - Lac Operon - submit questions I. Background: For the last week you explored the functioning of the enzyme

More information

HiPer Ion Exchange Chromatography Teaching Kit

HiPer Ion Exchange Chromatography Teaching Kit HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for

More information

Transformation Kit BACTERIAL TRANSFORMATION: GREEN FLUORESCENT PROTEIN. Partnership for Biotechnology and Genomics Education

Transformation Kit BACTERIAL TRANSFORMATION: GREEN FLUORESCENT PROTEIN. Partnership for Biotechnology and Genomics Education Transformation Kit BACTERIAL TRANSFORMATION: GREEN FLUORESCENT PROTEIN Partnership for Biotechnology and Genomics Education Barbara Soots Linda Curro Education Coordinator University of California Davis

More information

ANTIBIOTIC INHIBITION OF BACTERIA

ANTIBIOTIC INHIBITION OF BACTERIA ANTIBIOTIC INHIBITION OF BACTERIA STANDARDS 3.2.10B, 3.2.12B Apply process knowledge and evaluate experimental information 3.3.10B, 3.3.12B Chemical and structural basis of living organisms Westminster

More information

TransIT -2020 Transfection Reagent

TransIT -2020 Transfection Reagent Quick Reference Protocol, MSDS and Certificate of Analysis available at mirusbio.com/5400 INTRODUCTION TransIT -2020 Transfection Reagent is a high-performance, animal-origin free, broad spectrum reagent

More information

UltraClean Soil DNA Isolation Kit

UltraClean Soil DNA Isolation Kit PAGE 1 UltraClean Soil DNA Isolation Kit Catalog # 12800-50 50 preps New improved PCR inhibitor removal solution (IRS) included Instruction Manual (New Alternative Protocol maximizes yields) Introduction

More information

Catalase. ***You will be working with hot water, acids and bases in this laboratory*** ****Use Extreme Caution!!!****

Catalase. ***You will be working with hot water, acids and bases in this laboratory*** ****Use Extreme Caution!!!**** AP BIOLOGY BIOCHEMISTRY ACTIVITY #9 NAME DATE HOUR CATALASE LAB INTRODUCTION Hydrogen peroxide (H 2 O 2 ) is a poisonous byproduct of metabolism that can damage cells if it is not removed. Catalase is

More information

Name Class Date. Figure 13 1. 2. Which nucleotide in Figure 13 1 indicates the nucleic acid above is RNA? a. uracil c. cytosine b. guanine d.

Name Class Date. Figure 13 1. 2. Which nucleotide in Figure 13 1 indicates the nucleic acid above is RNA? a. uracil c. cytosine b. guanine d. 13 Multiple Choice RNA and Protein Synthesis Chapter Test A Write the letter that best answers the question or completes the statement on the line provided. 1. Which of the following are found in both

More information

INTERFERin in vitro sirna/mirna transfection reagent PROTOCOL. 1 Standard sirna transfection of adherent cells... 2

INTERFERin in vitro sirna/mirna transfection reagent PROTOCOL. 1 Standard sirna transfection of adherent cells... 2 INTERFERin in vitro sirna/mirna transfection reagent PROTOCOL DESCRIPTION INTERFERin is a powerful sirna/mirna transfection reagent that ensures efficient gene silencing and reproducible transfection in

More information

RNA & Protein Synthesis

RNA & Protein Synthesis RNA & Protein Synthesis Genes send messages to cellular machinery RNA Plays a major role in process Process has three phases (Genetic) Transcription (Genetic) Translation Protein Synthesis RNA Synthesis

More information

LAB 16 Rapid Colony Transformation of E. coli with Plasmid DNA

LAB 16 Rapid Colony Transformation of E. coli with Plasmid DNA LAB 16 Rapid Colony Transformation of E. coli with Plasmid DNA Objective: In this laboratory investigation, plasmids containing fragments of foreign DNA will be used to transform Escherichia coli cells,

More information

Transformation Protocol

Transformation Protocol To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic

More information

User Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube

User Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube User Manual CelluLyser Lysis and cdna Synthesis Kit Version 1.4 Oct 2012 From cells to cdna in one tube CelluLyser Lysis and cdna Synthesis Kit Table of contents Introduction 4 Contents 5 Storage 5 Additionally

More information

HCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise:

HCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise: HCS604.03 Exercise 1 Dr. Jones Spring 2005 Recombinant DNA (Molecular Cloning) exercise: The purpose of this exercise is to learn techniques used to create recombinant DNA or clone genes. You will clone

More information

Translation. Translation: Assembly of polypeptides on a ribosome

Translation. Translation: Assembly of polypeptides on a ribosome Translation Translation: Assembly of polypeptides on a ribosome Living cells devote more energy to the synthesis of proteins than to any other aspect of metabolism. About a third of the dry mass of a cell

More information

Molecular Genetics. RNA, Transcription, & Protein Synthesis

Molecular Genetics. RNA, Transcription, & Protein Synthesis Molecular Genetics RNA, Transcription, & Protein Synthesis Section 1 RNA AND TRANSCRIPTION Objectives Describe the primary functions of RNA Identify how RNA differs from DNA Describe the structure and

More information

How Does a Doctor Test for AIDS?

How Does a Doctor Test for AIDS? Edvo-Kit #S-70 How Does a Doctor Test for AIDS? S-70 Experiment Objective: The Human Immunodefi ciency Virus (HIV) is an infectious agent that causes Acquired Immunodefi ciency Syndrome (AIDS) in humans.

More information

BACTERIAL ENUMERATION

BACTERIAL ENUMERATION BACTERIAL ENUMERATION In the study of microbiology, there are numerous occasions when it is necessary to either estimate or determine the number of bacterial cells in a broth culture or liquid medium.

More information

Lecture 4. Polypeptide Synthesis Overview

Lecture 4. Polypeptide Synthesis Overview Initiation of Protein Synthesis (4.1) Lecture 4 Polypeptide Synthesis Overview Polypeptide synthesis proceeds sequentially from N Terminus to C terminus. Amino acids are not pre-positioned on a template.

More information

Transcription in prokaryotes. Elongation and termination

Transcription in prokaryotes. Elongation and termination Transcription in prokaryotes Elongation and termination After initiation the σ factor leaves the scene. Core polymerase is conducting the elongation of the chain. The core polymerase contains main nucleotide

More information

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage. 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert

More information

Structure and Function of DNA

Structure and Function of DNA Structure and Function of DNA DNA and RNA Structure DNA and RNA are nucleic acids. They consist of chemical units called nucleotides. The nucleotides are joined by a sugar-phosphate backbone. The four

More information

Protein Synthesis How Genes Become Constituent Molecules

Protein Synthesis How Genes Become Constituent Molecules Protein Synthesis Protein Synthesis How Genes Become Constituent Molecules Mendel and The Idea of Gene What is a Chromosome? A chromosome is a molecule of DNA 50% 50% 1. True 2. False True False Protein

More information

Transformation of E.coli with pgal

Transformation of E.coli with pgal The Biotechnology Education Company ATTENTION! This experiment includes either BactoBeads or LyphoCells. If you have received LyphoCells, please refer to the addendum posted on the last page of this literature.

More information

Luciferase Reporter Assay Kit User Manual

Luciferase Reporter Assay Kit User Manual Luciferase Reporter Assay Kit User Manual Cat. No. K2039-1 PT3392-1 (PR2Y278) Published 11/22/2002 Table of Contents I. Introduction 3 II. List of Components 5 III. Additional Materials Required 5 IV.

More information

Translation Study Guide

Translation Study Guide Translation Study Guide This study guide is a written version of the material you have seen presented in the replication unit. In translation, the cell uses the genetic information contained in mrna to

More information

BUGS" THAT PRODUCE DRUGS TO KILL "BUGS Microbes Produce Antibiotics

BUGS THAT PRODUCE DRUGS TO KILL BUGS Microbes Produce Antibiotics BUGS" THAT PRODUCE DRUGS TO KILL "BUGS Microbes Produce Antibiotics Science in the Real World Microbes In Action BUGS" THAT PRODUCE DRUGS TO KILL "BUGS is a curriculum unit developed as part of the Science

More information

Lecture Series 7. From DNA to Protein. Genotype to Phenotype. Reading Assignments. A. Genes and the Synthesis of Polypeptides

Lecture Series 7. From DNA to Protein. Genotype to Phenotype. Reading Assignments. A. Genes and the Synthesis of Polypeptides Lecture Series 7 From DNA to Protein: Genotype to Phenotype Reading Assignments Read Chapter 7 From DNA to Protein A. Genes and the Synthesis of Polypeptides Genes are made up of DNA and are expressed

More information

RIBOPROTECT. RNase Inhibitor RT33-020, RT33-100

RIBOPROTECT. RNase Inhibitor RT33-020, RT33-100 RIBOPROTECT RT33-020, RT33-100 RT33-020, RT33-100 RIBOPROTECT The RIBOPROTECT is a recombinant protein isolated and purified from Escherichia coli. It inhibits ribonuclease (RNase) activity of enzymes

More information

GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps)

GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) 1 GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) (FOR RESEARCH ONLY) Sample : Expected Yield : Endotoxin: Format : Operation Time : Elution Volume : 50-400 ml of cultured bacterial

More information

Enzymes: Practice Questions #1

Enzymes: Practice Questions #1 Enzymes: Practice Questions #1 1. Compound X increases the rate of the reaction below. Compound X is most likely A. an enzyme B. a lipid molecule C. an indicator D. an ADP molecule 2. The equation below

More information

UltraClean Forensic DNA Isolation Kit (Single Prep Format)

UltraClean Forensic DNA Isolation Kit (Single Prep Format) UltraClean Forensic DNA Isolation Kit (Single Prep Format) Catalog No. Quantity 14000-10 10 preps 14000-S 1 prep Instruction Manual Please recycle Version: 10302012 1 Table of Contents Introduction...

More information

The sequence of bases on the mrna is a code that determines the sequence of amino acids in the polypeptide being synthesized:

The sequence of bases on the mrna is a code that determines the sequence of amino acids in the polypeptide being synthesized: Module 3F Protein Synthesis So far in this unit, we have examined: How genes are transmitted from one generation to the next Where genes are located What genes are made of How genes are replicated How

More information

RT31-020 20 rxns. RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl

RT31-020 20 rxns. RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl Components RT31-020 20 rxns RT31-050 50 rxns RT31-100 100 rxns TRANSCRIPTME Enzyme Mix (1) 40 µl 2 x 50 µl 5 x 40 µl 2x RT Master Mix (2) 200 µl 2 x 250 µl 5 x 200 µl RNase H (E. coli) 20 µl 2 x 25 µl

More information

MGC premier Expression-Ready cdna clones TCH1103, TCM1104, TCR1105, TCB1106, TCH1203, TCM1204, TCR1205, TCB1206, TCH1303, TCM1304, TCR1305

MGC premier Expression-Ready cdna clones TCH1103, TCM1104, TCR1105, TCB1106, TCH1203, TCM1204, TCR1205, TCB1206, TCH1303, TCM1304, TCR1305 MGC premier Expression-Ready cdna clones TCH1103, TCM1104, TCR1105, TCB1106, TCH1203, TCM1204, TCR1205, TCB1206, TCH1303, TCM1304, TCR1305 The MGC premier Expression-Ready collection has the high quality,

More information

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280.

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. Technical Bulletin Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. PRINTED IN USA. Revised 4/06 AF9TB141 0406TB141 Wizard DNA Clean-Up System All technical literature is available on

More information

RT-PCR: Two-Step Protocol

RT-PCR: Two-Step Protocol RT-PCR: Two-Step Protocol We will provide both one-step and two-step protocols for RT-PCR. We recommend the twostep protocol for this class. In the one-step protocol, the components of RT and PCR are mixed

More information

An In-Gel Digestion Protocol

An In-Gel Digestion Protocol An In-Gel Digestion Protocol This protocol describes the digestion of a protein present in an SDS-PAGE gel band with trypsin. The band can be taken from either a 1D or 2D electrophoresis gel. Reagents

More information

Isolation of Starch degrading bacteria Enzymes in Action

Isolation of Starch degrading bacteria Enzymes in Action Isolation of Starch degrading bacteria Enzymes in Action Introduction In this laboratory exercise, you will be playing the role of biotechnologists in search of a new amylase. Since most industrially used

More information

Transcription: RNA Synthesis, Processing & Modification

Transcription: RNA Synthesis, Processing & Modification Transcription: RNA Synthesis, Processing & Modification 1 Central dogma DNA RNA Protein Reverse transcription 2 Transcription The process of making RNA from DNA Produces all type of RNA mrna, trna, rrna,

More information

www.biochemj.org/bj/330/0581/bj3300581.htm

www.biochemj.org/bj/330/0581/bj3300581.htm Ribosomes as Antibiotic Targets www.biochemj.org/bj/330/0581/bj3300581.htm Ware, Bioscience in the 21 st Century, 2009 PERSPECTIVE Widespread use of antibiotics after WWII improved human health globally

More information

Student Manual. pglo Transformation

Student Manual. pglo Transformation Student Manual pglo Transformation Lesson 1 Introduction to Transformation In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides

More information

Lab # 12: DNA and RNA

Lab # 12: DNA and RNA 115 116 Concepts to be explored: Structure of DNA Nucleotides Amino Acids Proteins Genetic Code Mutation RNA Transcription to RNA Translation to a Protein Figure 12. 1: DNA double helix Introduction Long

More information

THE ACTIVITY OF LACTASE

THE ACTIVITY OF LACTASE THE ACTIVITY OF LACTASE Lab VIS-8 From Juniata College Science in Motion Enzymes are protein molecules which act to catalyze the chemical reactions in living things. These chemical reactions make up the

More information

Catalytic Activity of Enzymes

Catalytic Activity of Enzymes Catalytic Activity of Enzymes Introduction Enzymes are biological molecules that catalyze (speed up) chemical reactions. You could call enzymes the Builders and Do-ers in the cell; without them, life could

More information

Quantifying Bacterial Concentration using a Calibrated Growth Curve

Quantifying Bacterial Concentration using a Calibrated Growth Curve BTEC 4200 Lab 2. Quantifying Bacterial Concentration using a Calibrated Growth Curve Background and References Bacterial concentration can be measured by several methods, all of which you have studied

More information

RiboZol RNA Extraction Reagents

RiboZol RNA Extraction Reagents RiboZol RNA Extraction Reagents Code Description Size N580-30ML-SAMPLE Ribozol TM RNA Extraction Reagent 30 ml N580-30ML Ribozol TM RNA Extraction Reagent 30 ml N580-100ML Ribozol TM RNA Extraction Reagent

More information

ab185915 Protein Sumoylation Assay Ultra Kit

ab185915 Protein Sumoylation Assay Ultra Kit ab185915 Protein Sumoylation Assay Ultra Kit Instructions for Use For the measuring in vivo protein sumoylation in various samples This product is for research use only and is not intended for diagnostic

More information

The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade plasmid DNA.

The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade plasmid DNA. INSTRUCTION MANUAL ZymoPURE Plasmid Gigaprep Kit Catalog Nos. D4204 (Patent Pending) Highlights The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade

More information

MTT Cell Proliferation Assay

MTT Cell Proliferation Assay ATCC 30-1010K Store at 4 C This product is intended for laboratory research purposes only. It is not intended for use in humans, animals or for diagnostics. Introduction Measurement of cell viability and

More information

LAB 11 PLASMID DNA MINIPREP

LAB 11 PLASMID DNA MINIPREP LAB 11 PLASMID DNA MINIPREP STUDENT GUIDE GOAL The objective of this lab is to perform extraction of plasmid DNA and analyze the results. OBJECTIVES After completion, the student should be able to: 1.

More information

CHAPTER 30: PROTEIN SYNTHESIS

CHAPTER 30: PROTEIN SYNTHESIS CHAPTER 30: PROTEIN SYNTHESIS (Translation) Translation: mrna protein LECTURE TOPICS Complexity, stages, rate, accuracy Amino acid activation [trna charging] trnas and translating the Genetic Code - Amino

More information

ExpressArt Bacterial H-TR cdna synthesis kit. With extreme selectivity against rrnas

ExpressArt Bacterial H-TR cdna synthesis kit. With extreme selectivity against rrnas ExpressArt Bacterial H-TR cdna synthesis kit With extreme selectivity against rrnas suitable for 2 to 4 µg total RNA Catalogue No. 8004-A30 (30 rxns) Reagents Materials are provided for 30 cdna synthesis

More information

BuccalAmp DNA Extraction Kit QuickExtract DNA Extraction Solution 1.0

BuccalAmp DNA Extraction Kit QuickExtract DNA Extraction Solution 1.0 QuickExtract DNA Extraction Solution 1.0 Cat. Nos. BQ0901S (CR, SC, RB, BS), BQ0908S (CR, SC, RB, BS), BQ0916S (CR, SC, RB, BS), QE09050, QEC091H, QEC89100, MB100BR, and MB100SP Please store the QuickExtract

More information

Lab Exercise 3: Media, incubation, and aseptic technique

Lab Exercise 3: Media, incubation, and aseptic technique Lab Exercise 3: Media, incubation, and aseptic technique Objectives 1. Compare the different types of media. 2. Describe the different formats of media, plate, tube etc. 3. Explain how to sterilize it,

More information

AP BIOLOGY 2009 SCORING GUIDELINES

AP BIOLOGY 2009 SCORING GUIDELINES AP BIOLOGY 2009 SCORING GUIDELINES Question 4 The flow of genetic information from DNA to protein in eukaryotic cells is called the central dogma of biology. (a) Explain the role of each of the following

More information

Transcription and Translation of DNA

Transcription and Translation of DNA Transcription and Translation of DNA Genotype our genetic constitution ( makeup) is determined (controlled) by the sequence of bases in its genes Phenotype determined by the proteins synthesised when genes

More information

Microbiology BIOL 275 DILUTIONS

Microbiology BIOL 275 DILUTIONS DILUTIONS Occasionally a solution is too concentrated to be used as is. For example, when one is performing manual blood counts, the blood contains too many cells to be counted as such. Or when performing

More information

Genomic DNA Extraction Kit INSTRUCTION MANUAL

Genomic DNA Extraction Kit INSTRUCTION MANUAL Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview

More information

PRODUCT INFORMATION...

PRODUCT INFORMATION... VIROMER RED In vitro plasmid DNA and mrna Standard Transfection PRODUCT INFORMATION... 2 GENERAL... 2 RED OR YELLOW?... 3 PROTOCOL GUIDELINES... 4 GENERAL REMARKS... 4 CELL CULTURE AND PLATING... 4 FORWARD/REVERSE

More information

CHAPTER 40 The Mechanism of Protein Synthesis

CHAPTER 40 The Mechanism of Protein Synthesis CHAPTER 40 The Mechanism of Protein Synthesis Problems: 2,3,6,7,9,13,14,15,18,19,20 Initiation: Locating the start codon. Elongation: Reading the codons (5 3 ) and synthesizing protein amino carboxyl.

More information

One Shot TOP10 Competent Cells

One Shot TOP10 Competent Cells USER GUIDE One Shot TOP10 Competent Cells Catalog Numbers C4040-10, C4040-03, C4040-06, C4040-50, and C4040-52 Document Part Number 280126 Publication Number MAN0000633 Revision A.0 For Research Use Only.

More information

Modeling DNA Replication and Protein Synthesis

Modeling DNA Replication and Protein Synthesis Skills Practice Lab Modeling DNA Replication and Protein Synthesis OBJECTIVES Construct and analyze a model of DNA. Use a model to simulate the process of replication. Use a model to simulate the process

More information

LAB 4. Cultivation of Bacteria INTRODUCTION

LAB 4. Cultivation of Bacteria INTRODUCTION LAB 4. Cultivation of Bacteria Protocols for use of cultivation of bacteria, use of general growth, enriched, selective and differential media, plate pouring, determination of temperature range for growth

More information

1.5 page 3 DNA Replication S. Preston 1

1.5 page 3 DNA Replication S. Preston 1 AS Unit 1: Basic Biochemistry and Cell Organisation Name: Date: Topic 1.5 Nucleic Acids and their functions Page 3 l. DNA Replication 1. Go through PowerPoint 2. Read notes p2 and then watch the animation

More information

4. DNA replication Pages: 979-984 Difficulty: 2 Ans: C Which one of the following statements about enzymes that interact with DNA is true?

4. DNA replication Pages: 979-984 Difficulty: 2 Ans: C Which one of the following statements about enzymes that interact with DNA is true? Chapter 25 DNA Metabolism Multiple Choice Questions 1. DNA replication Page: 977 Difficulty: 2 Ans: C The Meselson-Stahl experiment established that: A) DNA polymerase has a crucial role in DNA synthesis.

More information

DNA Replication & Protein Synthesis. This isn t a baaaaaaaddd chapter!!!

DNA Replication & Protein Synthesis. This isn t a baaaaaaaddd chapter!!! DNA Replication & Protein Synthesis This isn t a baaaaaaaddd chapter!!! The Discovery of DNA s Structure Watson and Crick s discovery of DNA s structure was based on almost fifty years of research by other

More information

Table of Contents. I. Description... 2. II. Kit Components... 2. III. Storage... 2. IV. 1st Strand cdna Synthesis Reaction... 3

Table of Contents. I. Description... 2. II. Kit Components... 2. III. Storage... 2. IV. 1st Strand cdna Synthesis Reaction... 3 Table of Contents I. Description... 2 II. Kit Components... 2 III. Storage... 2 IV. 1st Strand cdna Synthesis Reaction... 3 V. RT-PCR, Real-time RT-PCR... 4 VI. Application... 5 VII. Preparation of RNA

More information

DNA Isolation Kit for Cells and Tissues

DNA Isolation Kit for Cells and Tissues DNA Isolation Kit for Cells and Tissues for 10 isolations of 00 mg each for tissue or 5 x 10 7 cultured cells Cat. No. 11 81 770 001 Principle Starting material Application Time required Results Benefits

More information

a. Ribosomal RNA rrna a type ofrna that combines with proteins to form Ribosomes on which polypeptide chains of proteins are assembled

a. Ribosomal RNA rrna a type ofrna that combines with proteins to form Ribosomes on which polypeptide chains of proteins are assembled Biology 101 Chapter 14 Name: Fill-in-the-Blanks Which base follows the next in a strand of DNA is referred to. as the base (1) Sequence. The region of DNA that calls for the assembly of specific amino

More information

Bacterial Transformation and Plasmid Purification. Chapter 5: Background

Bacterial Transformation and Plasmid Purification. Chapter 5: Background Bacterial Transformation and Plasmid Purification Chapter 5: Background History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment

More information