LAB 7 DNA RESTRICTION for CLONING
|
|
- Duane Shelton
- 7 years ago
- Views:
Transcription
1 BIOTECHNOLOGY I DNA RESTRICTION FOR CLONING LAB 7 DNA RESTRICTION for CLONING STUDENT GUIDE GOALS The goals of this lab are to provide the biotech student with experience in DNA digestion with restriction enzymes for the purpose of gene cloning, and to teach about the structure and function of plasmid DNA. OBJECTIVES After completion, the student should be able to: 1. Define what a DNA restriction endonuclease is and how it functions to cut DNA 2. Estimate the size of the DNA bands that result from restriction digestion using gel electrophoresis. 3. Identify the various conformations of uncut plasmid DNA as seen in gel electrophoresis. 4. Discuss the different lifestyles of bacteriophage. 5. Estimate the size and concentration of plasmid DNA using gel electrophoresis results. BACKGROUND Plasmids Plasmids are circular pieces of DNA that exist independent of the chromosome of prokaryotic cells. Plasmids are retained because they carry genes that benefit the bacterium, such as genes for antibiotic resistance. Plasmid DNA exists naturally as a supercoiled molecule, in which the circle of DNA is folded upon itself, much like a twisted rubber band collapses from a circle to a twisted line (Figure 1a). When plasmids have nicks, or breaks along the sugar phosphate backbone of the DNA, the tension of coiling is relieved and a relaxed circular DNA results. The circle is no longer held together totally by covalent bonding between the sugars and phosphates, but instead is weakly held together by the hydrogen bonding between the base pairs. Plasmids also exist in another type of conformation that of interlocking rings called catenanes, which form during replication. There can be dimers (two rings), trimers (three rings), and tetramers (four rings). Each of the interlocked circles can be supercoiled (Figure 1a) or relaxed (Figure 1b). a. b. Figure 1. Plasmid conformation models; a. super coiled; b. relaxed dimer catenane Eilene Lyons Revised 1/12/
2 When uncut plasmid DNA is run on an agarose gel, many bands are usually visible. Recall that it is the size of the DNA that determines how quickly it can migrate through a gel in an electric field. The shape of the molecule also has a bearing on the migration the more compact the shape, the faster it will move. Each of the varying shapes that plasmid DNA can take allows it to move through agarose gel at many speeds. The supercoiled plasmids move fastest and are most abundant, catenanes move more slowly, and open circular or nicked DNA runs somewhere in between. Look for slight bands all along the path that the plasmid DNA travels in the gel. Figure 2 shows three conformation bands for an uncut 6.2 kb plasmid. Notice that the supercoiled band is most abundant (it is brighter, which indicates more DNA) and it runs at the same speed as a linear fragment of DNA that is just under 4 kb in size. Along with DNA, small fragments of transfer RNA and digested ribosomal and messenger RNA may be present. This is seen as a smear at the bottom of lane Wells Catenanes Nicked circles Supercoiled 6 kb 2 kb RNA Figure 2. Uncut plasmid DNA run on an agarose gel; lane 1 an uncut 6.2 kb plasmid; lane 2 kb Ladder standard DNA marker. Researchers make use of genetically engineered plasmids as cloning vectors. High copy number plasmids will replicate as many as times within each cell. Plasmids first engineered at the University of California bear the prefix puc and have been used widely in biotechnology research for the past 30 years. Cloning Basics The cloning of a gene first involves restriction enzyme digestion of chromosomal DNA. Restriction enzymes that cut less frequently are chosen, so that the resulting fragments are larger. The more nucleotides in the recognition sequence, the less frequently the sequence will occur, whereas the fewer the number of nucleotides, the more likely the sequence will occur, resulting in an increase in smaller fragments of DNA. Typically, a digestion reaction has a final volume of 20 l containing about 1 g of 7-2
3 plasmid DNA and at least 1 unit of restriction enzyme/ g of plasmid or phage DNA (about 5-10 units/ g for genomic DNA). Star activity, the indiscriminate cutting of DNA at sites other than the intended recognition sequences, can give abnormal results. Changes in the buffer cations (Mn 2+, Co 2+, instead of Mg 2+ ), the cation concentration, the ph of the solution, the presence of glycerol or ethanol in the reaction mixture and excess enzyme can all cause star activity resulting in more DNA fragments than expected. Once the DNA is cut properly, the restriction fragment containing the gene is identified, isolated and purified by a process referred to as Gene Clean. Fragments of the correct size are then joined, or ligated, with larger fragments of DNA called vectors, to create recombinant DNA. The vector DNA can be phage (bacteria-infecting virus), a plasmid, or a combination of the two (cosmids and phasmids). Once the fragments are ligated into the vector, bacterial cells are transformed by manipulations designed to take the recombinant DNA across cell membranes and into the cytoplasm of the cells. The transformation is plated on selective media and colonies that appear to have been transformed with the recombinant vector are cultured in liquid media overnight. During the growth of the bacterial culture, the gene of interest is cloned within the hundreds of plasmids replicated within each cell, giving enough DNA for further genetic procedures. The next step is restriction mapping to verify that the plasmid DNA truly contains the gene of interest. LABORATORY OVERVIEW In this lab a commercial plasmid will be digested with a restriction enzyme to serve as the vector for recombinant plasmid construction. In successive labs, a fragment of phage DNA will be isolated, purified and then joined to the commercial plasmid, forming a new recombinant plasmid. TIMELINE: Day 1 Set up digestions and run E-gels to confirm digestion; cast 0.8 % gel for the next lab, if time permits. SAFETY GUIDELINES Ethidium bromide is a strong mutagen. Gloves must always be worn when handling gels or buffers containing this chemical. Boiling agarose can cause burns. Wear hot gloves when removing agarose from hot plate or microwave oven. The electric current in a gel electrophoresis chamber is extremely dangerous. Never remove a lid or touch the buffer once the power is turned on. Make sure the counter where the gel is being run is dry. UV light, used to illuminate the DNA stained with ethidium bromide, is dangerous. Eye protection must be used. 7-3
4 MATERIALS - DNA Digestion puc 18 DNA, approximately [0.5 μg/μl]; dilute per instructor s directions Eco RI endonuclease 10x buffer for Eco RI, 50 µl/team Reaction grade dh 2 O Water bath set at 37 C automatic micropipetters and tips used tip container gloves Kim wipes 1.5 ml microcentrifuge tubes microcentrifuge tube rack Sharpie marking pen Floating microfuge tube racks for water bath MATERIALS - Gel Electrophoresis E-gels and rigs Uncut puc 18 DNA as control on gel Molecular Weight Marker DNA (with 10 L concentration labeled) Sharpie marking pen gloves dh 2 O automatic micropipetters and tips Used tip container Kim wipes 1.5 ml microcentrifuge tubes Microfuge tube rack UV Transilluminator UV Camera and film UV eye protection and face shields 7-4
5 PROCEDURE Each team will set up one digestion. Set the water bath at 37 C. PART I. DNA Digestion NOTE: We will be using commercial Lambda DNA cut with Eco RI, so only the puc 18 plasmid needs to be digested in this lab. 1. Completely thaw the restriction enzyme buffer. DO NOT USE BUFFERS THAT CONTAIN ICE; THE BUFFER CONCENTRATION WILL BE INCORRECT. 2. Add the following to a labeled 1.5 ml microcentrifuge tube to give a total volume of 30 l. Use a new tip for each reagent. (NOTE: A total volume of 30 L is used here so that part can be run on a gel now and the rest can be saved for the recombinant plasmid construction.) puc 18 DNA digest: Molecular grade dh 2 O puc 18 DNA ( 1.0 g) 10x buffer [1.0x final] *Eco RI (1U/ g of plasmid) Total volume: L L 3.0 L 0.5 L 30.0 l * Restriction enzymes are dissolved in glycerol, which is viscous. Take extra care to just touch the pipette tip to the surface, so none of the costly enzyme is wasted on the outside of the pipette tip. Let the micropipetter plunger up very slowly and hold the tip still to allow the glycerol to enter completely. Wipe the tip on the inside of the tube to save as much as possible, as most enzymes are very expensive. If glycerol appears on the outside of the tip, use a Kim wipe to remove it to prevent Star activity. 3. Spin briefly to mix. Incubate at 37 C in a water bath for at least 1 hour to overnight. (NOTE: While incubating, pour the gel you will use for the next lab when you perform the gene clean.) 4. Make a table of the expected results from the digestion of puc 18 and Lambda DNA + Eco RI (even though you will not cut Lambda in this lab). Include this table in the Analysis section of this lab in your lab notebook. (See the sample data table and the sample gel, below, and the maps and information at the end of this lab.) Table 1. Expected EcoR I Restriction Results DNA Size expected - uncut Sizes expected cut with EcoR I puc 18 plasmid Lambda DNA genome 7-5
6 Figure 3. Sample Gel of Expected Results Lane 1. Mol. Wt. Marker Lane 2. Uncut Lambda Lane 3. Lambda + EcoR I Lane 4. Uncut puc 18 Lane 5. puc 18 + EcoR I PART II. Casting a 0.8% Gel for DNA Electrophoresis and Gene Clean (next lab) 1. Weigh out 0.4 grams of agarose and place in a 250 ml flask. Add 50 ml 1X TAE buffer and microwave until completely melted, 1-2 minutes. 2. Set up the electrophoresis chambers as in previous lab. 3. When you can hold your hand on the bottom of the flask of heated agarose for 30 seconds, it is cool enough to pour. Check the volume after microwaving and add more dh 2 O to return the volume to 50 ml, if necessary. Pour the melted agarose into the casting tray. Insert the combs at the end that will have the negative electrode attached. (To make this determination, fit the lid on the gel box and connect the electrodes to the power supply.) 4. Allow the gel to cool and solidify (about minutes). Do not disturb the gel during this time. 5. When the agarose has solidified, gently remove the comb, pulling it straight upwards, in one motion. Wrap the gel in plastic wrap and/or place it in a zip lock bag that you have labeled with your group number and date. Store the gel at 4 C until the next lab. 7-6
7 Part III. Preparation of puc 18 digestions, gel loading and running 1. Add 15 µl of molecular grade water to a labeled 1.5 µl microcentrifuge tube. Pulse spin the digested puc 18 DNA in a microcentrifuge to pull any condensation to the bottom of the tube. Transfer 5 L of this digestion into the labeled microfuge tube. Place the remainder of the digestion in the class freezer box. 2. Add 10 L of molecular grade water to a fresh tube labeled as Marker DNA. Transfer 10 µl of the molecular weight marker DNA to this tube. (The first group to this point must perform the next step dilution of the control uncut puc 18. Dilute so that there is approximately 50 ng/20 ul for each E-gel to be run.) 3. Heat all puc 18 DNA restriction digestions at 65 C for 2 minutes. (NOTE: Determine if the molecular weight marker being used also must be heated prior to loading; if so, heat along with the DNA restrictions.) Pulse spin and place on ice. Make a note of the marker DNA you are using. Include a photocopy of information on this marker in your notebook. 4. Insert the E-gel into the holder and run for 2 minutes with the comb still in the wells to activate the ethidium bromide. The prerun is started by pressing down on either start button and holding until there is a beep. The gel rig will signal at the end of the 2 minute prerun. Remove the comb. 5. Do not leave any wells empty load each with a DNA sample or water. Load the DNA onto the E-gel(s) in the order decided on for the class. 6. Run the gel for 30 minutes, view on the transilluminator, and document for your lab notebook. 7. Before leaving the lab, make sure labels on all tubes are complete. The remainder of each digestion will be placed in the class freezer box and stored at -20 C to be used in the following labs. DATA ANALYSIS Determine if the puc 18 DNA is cut completely. If not, further incubation should yield complete cutting. The size of the DNA fragments can be estimated by comparing the distance each fragment has migrated relative to the molecular weight marker bands. Size determination has a 10% error, so remember that this is an estimate. (Do not graph the results just estimate by observing the molecular weight marker and the uncut puc 18 bands on the gel photo.) 7-7
8 QUESTIONS 1. What is the function of a plasmid and where are they found naturally? 2. Where do DNA restriction enzymes exist naturally and what is their function? 3. Explain why you would cut with a 6-base cutter rather than a 4-base cutter if you were planning to clone a gene from chromosomal DNA. 4. What is Star Activity? 5. What are the three conformations of uncut plasmid DNA that might be seen on a gel? 6. Was the DNA in your digestion completely cut? How did you know whether it was or was not cut? Size = 48,502 bp DNA MAPS Lambda DNA cut with EcoR I gel electrophoresis results: Sources:
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 informationRESTRICTION ENZYME ANALYSIS OF DNA
University of Massachusetts Medical School Regional Science Resource Center SUPPORTING MATHEMATICS, SCIENCE AND TECHNOLOGY EDUCATION 222 Maple Avenue, Stoddard Building Shrewsbury, MA 01545-2732 508.856.5097
More informationTransformation 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 informationDNA Electrophoresis Lesson Plan
DNA Electrophoresis Lesson Plan Primary Learning Outcomes: Students will learn how to properly load a well in an agarose gel. Students will learn how to analyze the results of DNA electrophoresis. Students
More informationThe Techniques of Molecular Biology: Forensic DNA Fingerprinting
Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins
More informationTroubleshooting Guide for DNA Electrophoresis
Troubleshooting Guide for Electrophoresis. ELECTROPHORESIS Protocols and Recommendations for Electrophoresis electrophoresis problem 1 Low intensity of all or some bands 2 Smeared bands 3 Atypical banding
More informationDNA: A Person s Ultimate Fingerprint
A partnership between the UAB Center for Community Outreach Development and McWane Center DNA: A Person s Ultimate Fingerprint This project is supported by a Science Education Partnership Award (SEPA)
More informationHiPer 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 informationLab 5: DNA Fingerprinting
Lab 5: DNA Fingerprinting You are about to perform a procedure known as DNA fingerprinting. The data obtained may allow you to determine if the samples of DNA that you will be provided with are from the
More informationELUTION OF DNA FROM AGAROSE GELS
ELUTION OF DNA FROM AGAROSE GELS OBTECTIVE: To isolate specific bands or regions of agarose-separated DNA for use in subsequent experiments and/or procedures. INTRODUCTION: It is sometimes necessary to
More informationTransformAid 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 informationGreen 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 informationLAB 14 DNA Restriction Analysis
Name: AP Biology Lab 14 LAB 14 DNA Restriction Analysis Introduction: DNA restriction analysis is at the heart of recombinant DNA technology and of the laboratories in this course. The ability to cut DNA
More informationAGAROSE GEL ELECTROPHORESIS:
AGAROSE GEL ELECTROPHORESIS: BEST PRACTICES (BACK TO THE BASICS) Unit of Tropical Laboratory Medicine April 2009 Marcella Mori WORKFLOW OF AGAROSE GEL ELECTROPHORESIS: THREE STEPS Agarose gel electrophoresis
More informationComputer 6B. Forensic DNA Fingerprinting
Forensic DNA Fingerprinting Computer 6B Scientists working in forensic labs are often asked to perform DNA profiling or fingerprinting to analyze evidence in law enforcement, mass disasters, and paternity
More informationTransformation 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 informationBacterial 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 informationAgarose Gel Electrophoresis with Food Color- Teacher Guide
Page 1 of 7 Project Home Gateway to the Project Laboratory Activities What the Project can do in the classroom Biotechnology Resources Favorite resources online and in print Agarose Gel Electrophoresis
More informationCloning GFP into Mammalian cells
Protocol for Cloning GFP into Mammalian cells Studiepraktik 2013 Molecular Biology and Molecular Medicine Aarhus University Produced by the instructors: Tobias Holm Bønnelykke, Rikke Mouridsen, Steffan
More informationObjectives: Vocabulary:
Introduction to Agarose Gel Electrophoresis: A Precursor to Cornell Institute for Biology Teacher s lab Author: Jennifer Weiser and Laura Austen Date Created: 2010 Subject: Molecular Biology and Genetics
More informationPlant Genomic DNA Extraction using CTAB
Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however
More informationCLONING 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 informationRAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis
RAINBOW ELECTROPHORESIS 1 An Introduction to Gel Electrophoresis INTRODUCTION This laboratory will demonstrate the basics of electrophoresis and the theory behind the separation of molecules on an agarose
More informationCrime Scenes and Genes
Glossary Agarose Biotechnology Cell Chromosome DNA (deoxyribonucleic acid) Electrophoresis Gene Micro-pipette Mutation Nucleotide Nucleus PCR (Polymerase chain reaction) Primer STR (short tandem repeats)
More informationSouthern Blot Analysis (from Baker lab, university of Florida)
Southern Blot Analysis (from Baker lab, university of Florida) DNA Prep Prepare DNA via your favorite method. You may find a protocol under Mini Yeast Genomic Prep. Restriction Digest 1.Digest DNA with
More informationA STUDY ON THE EFFECTIVENESS OF PEER TUTORING AS A TEACHING METHOD IN HIGH SCHOOL BIOTECHNOLOGY LABS. June Camerlengo. Santa Fe High School
A STUDY ON THE EFFECTIVENESS OF PEER TUTORING AS A TEACHING METHOD IN HIGH SCHOOL BIOTECHNOLOGY LABS. 1 June Camerlengo Santa Fe High School A STUDY ON THE EFFECTIVENESS OF PEER TUTORING AS A TEACHING
More informationrestriction enzymes 350 Home R. Ward: Spring 2001
restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually
More informationSTA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR)
STA DARD OPERATI G PROCEDURE FOR THE DETECTIO OF AFRICA SWI E FEVER VIRUS (ASFV) BY CO VE TIO AL POLYMERASE CHAI REACTIO (PCR) jmvizcaino@vet.ucm.es Av/ Puerta de Hierro s/n. 28040 Madrid. Tel: (34) 913944082
More informationBio 6 Restriction Enzyme Digestion Lab
Bio 6 Restriction Enzyme Digestion Lab Objectives Upon completion of this laboratory you will understand how to: 1) set up and carry out a restriction enzyme digest of DNA, 2) carry out agarose gel electrophoresis
More informationDNA Fingerprinting. Biotechnology - Electrophoresis & DNA Fingerprinting Biology 100 - Concepts of Biology 8.1. Name Instructor Lab Section.
Biotechnology - Electrophoresis & DNA Fingerprinting Biology 100 - Concepts of Biology 8.1 Name Instructor Lab Section Objectives: To gain a better understanding of: Fundamental Biotechnology Techniques
More informationRecombinant DNA & Genetic Engineering. Tools for Genetic Manipulation
Recombinant DNA & Genetic Engineering g Genetic Manipulation: Tools Kathleen Hill Associate Professor Department of Biology The University of Western Ontario Tools for Genetic Manipulation DNA, RNA, cdna
More informationLab 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 informationHCS604.03 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 informationDNA Technology Mapping a plasmid digesting How do restriction enzymes work?
DNA Technology Mapping a plasmid A first step in working with DNA is mapping the DNA molecule. One way to do this is to use restriction enzymes (restriction endonucleases) that are naturally found in bacteria
More informationGENETIC 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 informationTHE 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 informationActivity Sheets Enzymes and Their Functions
Name: Date: Activity Sheets Enzymes and Their Functions amylase What are Enzymes? starch glucose Enzymes are compounds that assist chemical reactions by increasing the rate at which they occur. For example,
More informationForensic DNA Fingerprinting Kit. Instruction Manual
Biotechnology Explorer Forensic DNA Fingerprinting Kit Instruction Manual Catalog #166-0007EDU explorer.bio-rad.com The kit is shipped at room temperature. Open immediately upon arrival and store reagent
More informationDNA Scissors: Introduction to Restriction Enzymes
DNA Scissors: Introduction to Restriction Enzymes Objectives At the end of this activity, students should be able to 1. Describe a typical restriction site as a 4- or 6-base- pair palindrome; 2. Describe
More informationAgarose Gel Electrophoresis
Treseder Lab Protocol Molecular Techniques Rev. 08/2007 Agarose Gel Electrophoresis Introduction Agarose gel electrophoresis is a quick and easy molecular technique used to analyze and separate nucleic
More informationInvestigating a Eukaryotic Genome: Cloning and Sequencing a Fragment of Yeast DNA
Investigating a Eukaryotic Genome: Cloning and Sequencing a Fragment of Yeast DNA Credits: This lab was created by Sarah C.R. Elgin and developed and written by Kathleen Weston-Hafer. Specific protocols
More informationCHEF Genomic DNA Plug Kits Instruction Manual
CHEF Genomic DNA Plug Kits Instruction Manual Catalog Numbers 170-3591 170-3592 170-3593 For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723) Table of
More informationSection IV: Detection and Sizing of DNA in Agarose Gels
Section IV: In This Section Guide to Lonza Ladders and Markers 96 Detecting DNA with GelStar and SYBR Green Nucleic Acid Gel Stains 98 Detecting DNA with Ethidium Bromide 102 High Sensitivity Detection
More informationTransformation 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 informationThe Analysis of Food Samples for the Presence of Genetically Modified Organisms
The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 5 Agarose Gel Electrophoresis M. Somma, M. Querci WORLD HEALTH ORGANIZATION REGIONAL OFFICE FOR EUROPE ORGANISATION
More informationRAGE. Plugs for RAGE/PFGE
1 RAGE Rotating Field Gel Electrophoresis (RAGE) is a variation on Pulsed Field Gel Electrophoresis (PFGE) and gives similar results. We use equipment that was only briefly marketed by Stratagene, at far
More informationFactors Affecting Enzyme Activity
INTRODUCTION Factors Affecting Enzyme Activity The chemical reactions occurring in living things are controlled by enzymes. An enzyme is a protein in the cell which lowers the activation energy of a catalyzed
More informationThe E. coli Insulin Factory
The E. coli Insulin Factory BACKGROUND Bacteria have not only their normal DNA, they also have pieces of circular DNA called plasmids. Plasmids are a wonderfully ally for biologists who desire to get bacteria
More informationRecombinant DNA and Biotechnology
Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study
More informationTroubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid
Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid Lina Jew Department of Microbiology & Immunology, University of
More informationGel Electrophoresis Teacher Instructions Suggested Grade Level: Grades 7-14 Class Time Required: 1 period (50 minutes)
Biological Sciences Initiative HHMI Gel Electrophoresis Teacher Instructions Suggested Grade Level: Grades 7-14 Class Time Required: 1 period (50 minutes) EQUIPMENT AND MATERIALS NEEDED (per group) Electrophoresis
More informationLAB 10 DNA TRANSFORMATION
LAB 10 DNA TRANSFORMATION STUDENT GUIDE GOAL The objective of this lab is to successfully perform DNA transformation of a recombinant plasmid and use blue-white selection to select recombinant clones.
More informationDNA SPOOLING 1 ISOLATION OF DNA FROM ONION
DNA SPOOLING 1 ISOLATION OF DNA FROM ONION INTRODUCTION This laboratory protocol will demonstrate several basic steps required for isolation of chromosomal DNA from cells. To extract the chromosomal DNA,
More informationMICB ABI PRISM 310 SEQUENCING GUIDE SEQUENCING OF PLASMID DNA
Plasmid DNA Preparation MICB ABI PRISM 310 SEQUENCING GUIDE SEQUENCING OF PLASMID DNA Introduction: I have always used the classic Alkaline Lysis miniprep method to isolate plasmid DNA. (See below) If
More information2D gel Protocol. 2. Determining Protein Concentration of cell lysates
2D gel Protocol 1. Lysis and Protein Extraction from cells Prepare cell lysates with Trizol extraction by following Kathleen Lyons s protocol: AfCS Procedure Protocol PP00000155, Version 1, 05/12/03 (Ref.1).
More informationPCR Optimization. Table of Contents Fall 2012
Table of Contents Optimizing the Polymerase Chain Reaction Introduction.....1 Review of Mathematics........ 3 Solving Problems of Dilution and Concentration: Two Approaches.. 4 Experiment Overview 7 Calculations
More informationAP BIOLOGY 2007 SCORING GUIDELINES
AP BIOLOGY 2007 SCORING GUIDELINES Question 4 A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to completion with two restriction enzymes in three separate treatments: EcoRI, HaeIII,
More informationHiPer Total RNA Extraction Teaching Kit
HiPer Total RNA Extraction Teaching Kit Product Code: HTBM012 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1 hour Agarose Gel Electrophoresis: 1 hour Storage Instructions:
More informationRT-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 informationPCR 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 informationPLB161A Laboratory XI a Genome Mapping
PLB161A Laboratory XI a Genome Mapping Restriction Digests and Agarose Gel Electrophoresis of Genomic DNA. A. Restriction Digests. Introduction Restriction enzymes are a class of DNA endonucleases, which
More informationLAB 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 informationDNA quality: electrophoresis, spectrophotometry and fluorometry
DNA quality: electrophoresis, spectrophotometry and fluorometry Ambika B Gaikwad ambika@nbpgr.ernet.in After isolation of DNA, quantification and analysis of quality are necessary to ascertain the approximate
More informationGROWING 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 informationStructure 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 informationDenaturing Gradient Gel Electrophoresis (DGGE)
Laboratory for Microbial Ecology Department of Earth, Ecological and Environmental Sciences University of Toledo Denaturing Gradient Gel Electrophoresis (DGGE) Background information Denaturing gradient
More informationBiotechnology Explorer
Biotechnology Explorer Restriction Digestion and Analysis of Lambda DNA Kit Instruction Manual Catalog #166-0002EDU explorer.bio-rad.com The kit is packaged and shipped as two modules. Open the modules
More informationDNA Separation Methods. Chapter 12
DNA Separation Methods Chapter 12 DNA molecules After PCR reaction produces many copies of DNA molecules Need a way to separate the DNA molecules from similar sized molecules Only way to genotype samples
More informationSDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies
SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies Pouring the resolving gel 1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and
More informationEnzymes: 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 informationHow To Test For Crime Scene Patterns
Biotechnology Explorer Forensic DNA Fingerprinting Kit Instruction Manual Catalog #166-0077EDU The kit is shipped at room temperature. Open immediately upon arrival and store reagent bag at 20 C within
More informationPyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-)
PyroPhage 3173 DNA Polymerase, Exonuclease Minus (Exo-) FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608)
More informationEffects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual
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
More informationCHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA
CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the
More informationWizard SV Gel and PCR Clean-Up System
TECHNICAL BULLETIN Wizard SV Gel and PCR Clean-Up System Instruc ons for Use of Products A9280, A9281, A9282 and A9285 Revised 12/10 TB308 Wizard SV Gel and PCR Clean-Up System All technical literature
More informationNorthern blot analysis for microrna. (Narry Kim s lab)
Northern blot analysis for microrna (Narry Kim s lab) Materials 1. 10~50 μg of total RNA extracted from HeLa cells treated with sirna 2. RNA loading buffer 3. Probe: DNA oligonucleotide complementary to
More informationReduced Representation Bisulfite Sequencing for Methylation Analysis Preparing Samples for the Illumina Sequencing Platform
Reduced Representation Bisulfite Sequencing for Methylation Analysis Preparing Samples for the Illumina Sequencing Platform Introduction, 3 Sample Prep Workflow, 4 Best Practices, 5 DNA Input Recommendations,
More informationABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S.
A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA... A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA from Shed Reptile Skin ABSTRACT
More informationLecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology
Lecture 13: DNA Technology DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology DNA Sequencing determine order of nucleotides in a strand of DNA > bases = A,
More informationBiotechnology Explorer
Biotechnology Explorer Analysis of Precut Lambda DNA Kit Instruction Manual Catalog #166-0001EDU explorer.bio-rad.com Ships at room temperature. Store DNA in the refrigerator (4 C) or freezer ( 20 C) within
More informationGRS 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 informationSection III: Loading and Running DNA in Agarose Gels
Section III: In This Section DNA Loading 90 Loading Buffers 91 Optimal Voltage and Electrophoretic Times 92 Fast Running Protocols for High Resolution in MetaPhor Agarose Gels 93 References 94 89 Section
More informationProtocol Micro Lab. K:\Microlab_protocols\Protocols_active\03 Instrument manuals\ml03_001_001 DGGE.doc. Preparation of gels
Preparation of gels Cautions: Wear gloves all times when handling acryl amide and form amide solutions and all work with acryl amide and form amide has to be done in a flow hood. Acryl amide is very toxic.
More informationExpressArt 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 informationIntroduction to cloning
1 of 14 Introduction to cloning Aim The aim of this protocol is to serve as a general guideline to mainstream molecular cloning of Gene of Interest ( GOI ). Overview GOI Sequence Transformation into Bacteria
More informationApplication Guide... 2
Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied
More informationTaq98 Hot Start 2X Master Mix
Taq98 Hot Start 2X Master Mix Optimized for 98C Denaturation Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608) 831-9011 FAX: (608) 831-9012 lucigen@lucigen.com
More informationHow Does a Genetic Counselor Detect Mutant Genes? SECTION E. How Genes and the Environment Influence Our Health CHAPTER 3
CHAPTER 3 How Genes and the Environment Influence Our Health SECTION E How Does a Genetic Counselor Detect Mutant Genes? Chapter 3 Modern Genetics for All Students T 211 Chapter 3: Section E Background
More informationLab # 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 informationNimbleGen DNA Methylation Microarrays and Services
NimbleGen DNA Methylation Microarrays and Services Sample Preparation Instructions Outline This protocol describes the process for preparing samples for NimbleGen DNA Methylation microarrays using the
More informationBiology 3A Laboratory: Enzyme Function
Biology 3A Laboratory: Enzyme Function Objectives To be able to list the general characteristics of enzymes. To study the effects of enzymes on the rate of chemical reactions. To demonstrate the effect
More informationThe 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 information50 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 informationDNA Fingerprinting. Unless they are identical twins, individuals have unique DNA
DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence
More informationAn 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 informationProcedure for RNA isolation from human muscle or fat
Procedure for RNA isolation from human muscle or fat Reagents, all Rnase free: 20% SDS DEPC-H2O Rnase ZAP 75% EtOH Trizol Chloroform Isopropanol 0.8M NaCitrate/1.2M NaCl TE buffer, ph 7.0 1. Homogenizer-probe
More informationCHEM 2423 Recrystallization of Benzoic Acid EXPERIMENT 4 - Purification - Recrystallization of Benzoic acid
EXPERIMENT 4 - Purification - Recrystallization of Benzoic acid Purpose: a) To purify samples of organic compounds that are solids at room temperature b) To dissociate the impure sample in the minimum
More informationEXTRACTION OF DNA FROM CALF THYMUS CELLS Revised 2/1/96 Introduction
Revised 2/1/96 Introduction Cells may be classified into two primary types depending on whether they have a discrete nucleus (eukaryotic) or do not (prokaryotic). Prokaryotes include bacteria, such as
More informationOne 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 informationUse of Micropipettes
Use of Micropipettes Prior to lab you should understand: The function of micropipettes in the laboratory Basic parts of micropipette What volumes are measured with P, P and P1 micopipettors How to read
More information