This article reprinted from: Dooley, M. M Restriction endonuclease digestion of a plasmid.
|
|
- Dorothy Lawson
- 6 years ago
- Views:
Transcription
1 This article reprinted from: Dooley, M. M Restriction endonuclease digestion of a plasmid. Pages , in Tested Studies for Laboratory Teaching, Volume 29 (K.L. Clase, Editor). Proceedings of the 29th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 433 pages. Compilation copyright 2008 by the Association for Biology Laboratory Education (ABLE) ISBN All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner. Use solely at one s own institution with no intent for profit is excluded from the preceding copyright restriction, unless otherwise noted on the copyright notice of the individual chapter in this volume. Proper credit to this publication must be included in your laboratory outline for each use; a sample citation is given above. Upon obtaining permission or with the sole use at one s own institution exclusion, ABLE strongly encourages individuals to use the exercises in this proceedings volume in their teaching program. Although the laboratory exercises in this proceedings volume have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in this volume. The focus of ABLE is to improve the undergraduate biology laboratory experience by promoting the development and dissemination of interesting, innovative, and reliable laboratory exercises. Visit ABLE on the Web at:
2 Restriction Endonuclease Digestion of a Plasmid Margaret M. Dooley Biology Department College of Staten Island The City University of New York 2800 Victory Boulevard, Staten Island, NY dooley@mail.csi.cuny.edu Abstract: Recombinant DNA technology is widely utilized and therefore, undergraduate laboratory courses incorporate these techniques. This reliable laboratory exercise introduces the student to plasmid vectors, restriction enzymes, and agarose gel electrophoresis. Restriction digestion of pbr325 with PstI and HindIII, in single and double digests, was performed and analyzed on an agarose gel. Students constructed a standard curve based on the migration of fragments from a HindIII digest of bacteriophage λ DNA and used that curve to estimate the sizes of DNA fragments in the pbr325 digests. In this exercise, measurements on the largest λ fragment were included to illustrate the resolution limits of agarose gels and the proper use of standard curves. Introduction The plasmid cloning vector, pbr325, carries genes coding resistance to the antibiotics tetracycline, ampicillin and chloramphenicol. Unique restriction sites are located in each antibiotic resistance gene, for example, PstI in ampicillin, BamHI in tetracycline and EcoRI in chloramphenicol (Bolivar, 1978; Prentki et al., 1981) and at other positions around the plasmid, e.g., HindIII which is located in the promoter for the tetracycline resistance gene (Rodriguez et al., 1979). The location of unique restriction sites at various positions around the plasmid permits the formation of easily visualized and well separated restriction fragments. Thus, this vector is well suited for use in instructional restriction analysis protocols. Using this vector also introduces the student to the structure and utilization of the widely utilized plasmid cloning vector pbr322 and its derivatives. Students digest pbr325 with HindIII and PstI, perform agarose gel electrophoresis, and estimate the fragment sizes from a standard curve generated from a λ HindIII digest. In this exercise, this plot includes the 23,130 base pair (bp) λ fragment to illustrate the limited resolution range of agarose gels and the necessity of having points on the standard curve bracket the experimental measurements. Materials and Methods Materials and Solutions Restriction enzymes, restriction buffers, and λ DNA were from New England BioLabs; pbr325 was from Sigma-Aldrich. Plasmid DNA was resuspended in sterile TE buffer to 0.1 µg/µl, and λ DNA to 0.4 µg/µl. Electrophoresis reagents (agarose, loading dye, Tris-Borate-EDTA (TBE) buffer, ethidium bromide solution) and Tris-EDTA buffer can be purchased from Carolina Biological Supply Company or prepared according to Sambrook et al. (1989). Tris-EDTA (TE) is
3 390 ABLE 2007 Proceedings Vol mM Tris-HCl (ph 8.0), 1mM EDTA (ph 8.0); 5X TBE stock solution is 54 g Tris base, 27.5 g boric acid, 20 ml 0.5 M EDTA (ph8.0); loading dye is 0.25% bromophenol blue, 0,25% xylene cyanol FF, 40% (w/v) sucrose in water (Sambrook et al., 1989). Safety Students were carefully supervised when they started the electrophoresis to prevent any safety hazards. Staining was with 1 µg/ml ethidium bromide which is a mutagen and suspect carcinogen. Therefore, to minimize risk, students were not allowed to handle the staining solution, aerosols were avoided, and a 5 mg/ml stock solution was purchased, not a powder. Ethidium bromide solution was chemically inactivated before disposal (Quillardet and Hofnung, 1988) or collected on commercially available filters which were discarded with hazardous waste. To protect the retina and skin from shorter wavelength ultraviolet light, an ultraviolet absorbing shield was always used when viewing gels. Experimental Procedure Students added all reagents as listed in Table 1 to digest plasmid (0.5 µg) with HindIII and PstI in both single and double digests. A HindIII digest of λ DNA (2 µg) was also performed to provide molecular weight standards on the agarose gel. After incubation for 1 hour at 37 C, samples were heated to 60 C for 3 minutes to melt the λ cohesive ends before loading on a 0.8% agarose gel and performing electrophoresis at volts for approximately 1 hour until the dye front was 1-2 cm from the end of the gel. If the experiment could not be completed in one laboratory session, samples were frozen after the restriction digestion and the 60 C incubation and electrophoresis was performed during a second session. The technician stained the gels in 1 µg/ml ethidium bromide for at least 1 hour and rinsed with water before viewing on an ultraviolet emitting transilluminator. The gels were photographed with Polaroid 667 film. The fragment migration distances were measured on an 8 X 11 inch print of the digitized photograph or alternatively, an expanded copy, or the Polaroid print itself. Table 1. Restriction reaction mixes Tube # Water Buffer DNA Enzyme 1 10 µl 4 µl 5X HindIII buffer 5 µl λ 1 µl HindIII 2 10 µl 4 µl 5X PstI buffer 5 µl plasmid 1 µl PstI 3 9 µl 4 µl 5X HindIII buffer 5 µl plasmid 1 µl PstI AND 1 µl HindIII 4 10 µl 4 µl 5X HindIII buffer 5 µl plasmid 1 µl HindIII 5 11 µl 4 µl 5X HindIII buffer 5 µl plasmid --- Results and Discussion The results of an ideal gel are shown in Figure 1A. Comparison of either single digest with the λ ladder confirms the size of pbr325 which has 5,995 base pairs (bp). The double digest
4 Poster Session 391 produces fragments that are approximately 2,400 and 3,600 bp as expected. Clearly, HindIII and PstI each cleave pbr325 only once and at different sites on the plasmid. Most undigested plasmid migrates closer to the wells than the linear pbr325 in the single digest lanes, confirming that both restriction enzymes cleaved pbr325 and emphasizing the need to compare like conformations in size determinations. 1A 1B Figure 1A. Agarose gel electrophoresis of restriction digests. Lane 1, λ DNA digested with HindIII (the size of each fragment is indicated in base pairs); lane 2, pbr325 digested with PstI; lane 3, pbr325 digested with PstI and HindIII; lane 4, pbr325 digested with HindIII; and lane 5, undigested pbr325. 1B. Standard curve for fragment size determination. The distance each λ HindIII fragment migrated was measured and plotted (arithmetic X-axis) verses the size in base pairs (logarithmic Y-axis).
5 392 ABLE 2007 Proceedings Vol. 29 Figure 1B shows the standard curve semilog plot that was used to determine the fragment sizes. A 0.8% gel cannot resolve fragments in the 23,130 bp range and therefore, this λ fragment is not on the linear portion of the plot which extends from 2,027 to at most 9,416 bp. This can be used to illustrate the importance of bracketing the unknown points with the standard curve as a linear relationship might not continue beyond the measured range. In the case of the λ HindIII ladder, if the 2,027-9,416 bp linear portion of the curve were incorrectly extrapolated beyond the highest point, the apparent size of the 23,130 bp fragment would be 12,000 bp, nearly a two fold error. Clearly, it is inappropriate to extrapolate a standard curve beyond the measured range. The 2,027-9,416 bp linear portion of the standard curve also reflects the fragment sizes that can be properly resolved; the steeper segment shows the size where the gel can no longer suitably separate fragments. The lack of resolution of larger DNA s can be emphasized by asking students to predict how far 23,000 and 22,700 bp fragments would migrate (both 62 mm) compared to the easily resolved 2,322 and 2,027 bp fragments (110 and 115 mm). Assessment was from grading of student lab reports and a quiz. In this exercise, students gained knowledge of the pbr325 cloning vehicle, the restriction digestion procedure, the agarose gel electrophoresis procedure, the interpretation of agarose gel patterns, and certain dilution procedures. The resolution limits of agarose gels and the correct use of standard curves were emphasized by considering the data on the largest λ fragment. In addition, students acquired the information and practice required to properly use micropipettors, microcentrifuges, and electrophoresis equipment. Literature Cited Bolivar, F Construction and Characterization of new cloning vehicles. Gene 4: Prenrki, P., F. Karch, S. Iida and J. Meyer The plasmid cloning vector pbr325 contains a 482 base pair long inverted duplication. Gene 14: Quillardet, P. and M. Hofnung Ethidium bromide and safety-readers suggest alternative solutions. (Letter to editor) Trends in Genetics 4: 89. Rodriguez, R. L., R. W. West, H. L. Heyneker, F. Bolivar, H. W. Boyer Characterizing wildtype and mutant promoters of the tetracycline resistance gene in pbr313. Nucleic Acids Research 6: Sambrook, J., E. F. Fritsch, and T. Maniatis Molecular Cloning A Laboratory Manual. Book 3. Second Edition. Cold Spring Harbor Laboratory Press, 1659 pages. About the Author Margaret M. Dooley earned her B.A. from Cornell University and her Ph.D. from Syracuse University. She received postdoctoral training at the University of California, Davis and Rutgers University before joining the faculty of the College of Staten Island where she teaches genetics, microbiology and general biology courses Margaret M. Dooley
6 Restriction Endonuclease Digestion of a Plasmid Margaret M. Dooley, Biology Department, College of Staten Island/The City University of New York, Staten Island, NY, dooley@mail.csi.cuny.edu Abstract Recombinant DNA technology is widely utilized and therefore, undergraduate laboratory courses incorporate these techniques. This reliable laboratory exercise introduces the student to plasmid vectors, restriction enzymes, and agarose gel electrophoresis. Restriction digestion of pbr325 with PstI and HindIII, in single and double digests, was performed and analyzed on an agarose gel. Students constructed a standard curve based on the migration of fragments from a HindIII digest of DNA and used that curve to estimate the sizes of DNA s in the pbr325 digests. Results from the gel were as expected: PstI and HindIII single digests produced one band at approximately 6000 bp confirming the size of pbr325 and the double digest produced two fragments approximately 2400 and 3600 bp. Clearly, these restriction endonucleases that cleave pbr325 only once are located at different positions on the plasmid. Questions to promote further thought and reinforce mathematics skills were included at the end of the exercise. In particular, the necessity of having points on the standard curve bracket the unknown data is emphasized by considering the apparent molecular weight of the 23,130 bp fragment. Fragments of this size cannot be properly resolved on this type of gel. If this band were of unknown size, extrapolation of the linear portion of the standard curve would estimate its size to be approximately 12,000 bp. Clearly, it is inappropriate to extrapolate a standard curve beyond the measured range. The linear portion of the standard curve also reflects the fragment sizes that can be properly resolved; the curved segment shows the size where the gel can no longer suitably separate fragments. Materials and Methods Materials and Solutions Restriction enzymes, restriction buffers, and DNA were from New England BioLabs; pbr325 was from Sigma-Aldrich. Plasmid DNA was resuspended in sterile TE buffer to 100 g/ml (0.1 g/ l) and DNA to 400 g/ml (0.4 g/ l). Electrophoresis reagents (agarose, loading dye, Tris-Borate-EDTA (TBE) buffer, ethidium bromide solution) and Tris-EDTA buffer can be purchased from Carolina Biological Supply Company or prepared according to Sambrook et al., Safety Students were carefully supervised when they started the electrophoresis to prevent any safety hazards. Staining was with 1 g/ml ethidium bromide which is a mutagen and suspect carcinogen. Therefore, to minimize risk, students were not allowed to handle the staining solution, aerosols were avoided and a 5 mg/ml stock solution was purchased, not a powder. Ethidium bromide solution was chemically inactivated before disposal (Quillardet, 1988) or collected on commercially available filters which were discarded with hazardous waste. To protect the retina and skin from shorter wavelength UV light, a UV absorbing shield was always used when viewing gels. Experimental Procedure Students added all reagents as listed in Table I to digest plasmid (0.5 g) with HindIII and PstI in both single and double digests. A HindIII digest of DNA (2 g) was also performed to provide molecular weight standards on the agarose gel. After incubation for 1 hour at 37 C, samples were heated to 60 C for 3 minutes to melt the cohesive ends before loading on a 0.8% agarose gel and performing electrophoresis at V approximately 1 hour until the dye front was 1-2 cm from the end of the gel. If the experiment could not be completed in one laboratory session, samples were frozen after the restriction digestion and the 60 C incubation and electrophoresis was performed during a second session. The technician stained the gels in 1 g/ml ethidium bromide for at least 1 hour and rinsed with water before photographing with Polaroid 667 film. The fragment migration distance was measured on a 8 X 11 print of the digitized photograph. Alternatively, an expanded copy or the Polaroid print could be measured. Assessment was from grading of student lab reports and a quiz that included questions about restriction digestion, use of plasmids in cloning experiments, agarose gel electrophoresis, and dilution of buffers. Students gained knowledge of (i) the specificity of restriction enzymes, (ii) the composition of restriction digestion reaction mixes, (iii) the agarose gel electrophoresis procedure, (iv) the interpretation of agarose gel patterns, (v) certain dilution procedures, and (vi) laboratory skills such as using micropipettors, microfuges, and electrophoresis equipment. Details of the student protocol and results will be presented. Introduction The plasmid cloning vector, pbr325, carries genes coding resistance to the antibiotics tetracycline, ampicillin and chloramphenicol. Unique restriction sites are located in each antibiotic resistance gene, for example, PstI in ampicillin, BamHI in tetracycline and EcoRI in chloramphenicol (Bolivar,1978; Prentki, 1981) and at other positions around the plasmid, e.g., HindIII which is located in the promoter for the tetracycline resistance gene (Rodriguez et al., 1979). The location of unique restriction sites at various positions around the plasmid permits the formation of easily visualized and well separated restriction fragments. Thus, this vector is well suited for use in instructional restriction analysis protocols. Using this vector also introduces the student to the structure and utilization of the widely utilized plasmid cloning vector pbr322 and its derivatives. Students digest pbr325 with HindIII and PstI, perform agarose gel electrophoresis, and estimate the fragment sizes from a standard curve generated from a HindIII digest. Results and Discussion The results of an ideal gel are shown in Figure 1. Comparison of either single digest with the ladder confirms the size of pbr325 which has 5995 bp. The double digest produces fragments that are approximately 2400 and 3600 bp as expected. Most undigested plasmid migrates closer to the wells than the linear pbr325 in the single digest lanes, confirming that both restriction enzymes cleaved pbr325 and emphasizing the need to compare like conformations in size determinations. Figure 2 shows the standard curve semilog plot that was used to determine the fragment sizes. A 0.8% gel cannot resolve fragments in the bp range and therefore, this fragment is not on the linear portion of the plot. This can be used to illustrate the importance of bracketing the unknown points with the standard curve as a linear relationship might not continue beyond the measured range. In the case of the HindIII ladder, if the linear portion of the curve were incorrectly extrapolated beyond the 9416 bp point, the apparent size of the bp fragment would be 12,000 bp, nearly a two fold error. Also, the lack of resolution of larger DNA s can be emphasized by asking students to predict how far and bp fragments would migrate (both 62 mm) compared to the easily resolved 2322 and 2027 bp fragments (110 and 115 mm). Thus, this exercise can be used to introduce standard curves, agarose gel electrophoresis, and restriction digestion. References Bolivar, F Construction and Characterization of new cloning vehicles Gene 4: Prenrki, P., F. Karch, S. Iida and J. Meyer The plasmid cloning vector pbr325 contains a 482 base-pair-long inverted duplication. Gene 14: Quillardet, P. and Hofnung Ethidium bromide and safety-readers suggest alternative solutions.(letter to editor) Trends in Genetics 4: 89. Rodriguez, R. L., West, R. W., Heyneker, H. L., Bolivar F, Boyer, H. W Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pbr313. Nucleic Acids Res. 6: Sambrook, J., E. F. Fritsch, and T. Maniatis Molecular Cloning A Laboratory Manual Second Edition. Cold Spring Harbor Laboratory Press
Agarose Gel Electrophoresis of DNA. By: Sahar alsubaie
Agarose Gel Electrophoresis of DNA By: Sahar alsubaie principle : Agarose Gel Electrophoresis uses electrical field to separate macromolecules (DNA and Protein) that differ in size, charge and configuration.
More information10 Restriction Analysis of Genomic DNA
10 Restriction Analysis of Genomic DNA Objectives: A) To determine the rough location of restriction sites of an unknown restriction enzyme and B) to use this information to determine the identity of this
More informationBIOL/CHEM 475 Spring 2007 RESTRICTION ENDONUCLEASES AND BACTERIOPHAGE λ
BIOL/CHEM 475 Spring 2007 RESTRICTION ENDONUCLEASES AND BACTERIOPHAGE λ Lambda (λ) is a temperate Escherichia coli bacteriophage. Optional read about phage λ: http://www.asm.org/division/m/fax/lamfax.html
More informationDNA Labeling Kits Fluorescein-dCTP and -dutp Instruction Manual
DNA Labeling Kits Fluorescein-dCTP and -dutp Instruction Manual Catalog Number 170-8223 170-8224 For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
More informationDNA Labeling Kits Texas Red -dctp and -dutp Instruction Manual
DNA Labeling Kits Texas Red -dctp and -dutp Instruction Manual Catalog Number 170-8221 170-8222 For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723) Bio-Rad
More informationITS Sequencing in Millepora. 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector
Page 1 of 5 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector 1. Digest 1 µg of pbluescript with Eco RI 2. Following digestion, add 0.1 volumes of 3M sodium acetate (ph
More informationLAB 6: Agarose Gel Electrophoresis of Restriction Digested Plasmid DNA
LAB 6: Agarose Gel Electrophoresis of Restriction Digested Plasmid DNA I. Objectives The purpose of today s lab is to learn how to set up and run an agarose gel, separate DNA fragments on the gel, and
More informationRecombination of Antibiotic Resistance Genes
LABORATORY 9 Recombination of Antibiotic Resistance Genes LABORATORY 9 BEGINS AN EXPERIMENTAL STREAM designed to construct and analyze a recombinant DNA molecule. The starting reagents are the relaxed
More informationDNA Visualizer Extraction Kit
DNA Visualizer Extraction Kit Catalog Number D0006 50 reactions Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 General Information...
More informationMission (Im)possible: Plasmid Mapping Student Materials
Mission (Im)possible: Plasmid Mapping Student Materials Introduction... 2 Pre-Lab Questions... 6 Lab Protocol... 7 Data Collection Worksheet... 11 Post-Lab Questions and Analysis... 12 Last updated: August
More informationComparing the Agilent 2100 Bioanalyzer Performance to Traditional DNA Analysis Techniques
Comparing the Agilent 2100 Bioanalyzer Performance to Traditional DNA Analysis Techniques Application Note Author Deborah Vitale Agilent Technologies, Inc. Palo Alto, CA, USA Abstract This Application
More informationUsing a Controlled Experiment to Identify Two Unknown Plasmids Edwin Braddy, River Ridge Middle/High School, New Port Richey, FL
INTRODUCTION To close the yellow note, click once to select it and then click the box in the upper left corner. To open the note, double click (Mac OS) or right click (Windows) on the note icon. Using
More informationIdentification of Unknown Plasmid Code Named 681A18
Identification of Unknown Plasmid Code Named 681A18 By Cody Latham Plasmids are small circular, doublestranded DNA molecules commonly found in bacteria that are separate from the chromosomal DNA found
More informationMission (Im)possible: Determine the Identity of Unknown Plasmids. Student Materials. Introduction Lab Protocol... 5
Mission (Im)possible: Determine the Identity of Unknown Plasmids Student Materials Introduction... 2 Lab Protocol... 5 Data Collection Worksheet... 9 Pre-Lab Questions... 10 Post-Lab Questions and Analysis...
More informationExercise 20 GEL ELECTROPHORESIS OF DNA SAMPLES (Plasmids, PCR products & Restriction Fragments)
Exercise 20 GEL ELECTROPHORESIS OF DNA SAMPLES (Plasmids, PCR products & Restriction Fragments) Introduction Gel electrophoresis is a technique or procedure allowing DNA fragments to be separated on the
More informationDNA RESTRICTION ANALYSIS
DNA RESTRICTION ANALYSIS In this experiment, DNA from the bacteriophage Lambda (48,502 base pairs in length) is cut with a variety of restriction enzymes and the resulting fragments are separated using
More informationMOLECULAR GENETICS: TRANSFORMATION AND CLONING adapted by Dr. D. L. Vogelien
Introduction MOLECULAR GENETICS: TRANSFORMATION AND CLONING adapted by Dr. D. L. Vogelien The field of molecular genetics has resulted in a number of practical applications that have been of tremendous
More informationManipulation of Purified DNA
Manipulation of Purified DNA To produce the recombinant DNA molecule, the vector, as well as the DNA to be cloned, must be cut at specific points and then joined together in a controlled manner by DNA
More information10X ligation buffer ligase 1 vector DNA insert DNA H 2 O. 10 µl Total Volume. 10X ligation buffer ligase 1 vector DNA insert DNA
Biol/Chem 475 S07 Study problems for quiz 1 See also questions posed in lab handouts including ligase handout Answers to questions 1&2 included at the end of this document. 1. You plan to clone a 1.0 kb
More informationAP Biology: Unit 5: Development. Forensic DNA Fingerprinting: Using Restriction Enzymes Bio-Rad DNA Fingerprinting Kit
Forensic DNA Fingerprinting: Using Restriction Enzymes Bio-Rad DNA Fingerprinting Kit Background: Scientists working in forensic labs are often asked to perform DNA profiling or fingerprinting to analyze
More informationAP Biology Lab 6 MOLECULAR BIOLOGY
AP Biology Laboratory Date: Name and Period: AP Biology Lab 6 MOLECULAR BIOLOGY OVERVIEW In this lab you will investigate some basic principles of molecular biology: 1. Plasmids containing specific fragments
More informationpamp, pkan, or pblu?
pamp, pkan, or pblu? Introduction In my final biotechnology lab project, I was given an unidentified plasmid, in which I was required to perform restriction digests using restriction enzymes. A plasmid,
More informationDNA Restriction Digestion Analysis
PR041 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DNA Restriction Digestion Analysis Teacher s Guidebook (Cat. # BE-307) think proteins!
More informationPrepared By: Mageswary Sivalingam
Prepared By: Mageswary Sivalingam A technique in which charged molecules are separated by their migration in an electric field. Applications - Gel electrophoresis is used in forensics, molecular biology,
More informationHow does electrophoresis work? The gel is made from agarose, DNA is a negative molecules, Molecules sort based on: Charge, Size, shape.
Lab six:. Gel Electrophoresis: What is Gel Electrophoresis? Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used
More informationObjectives Introduction restriction endonucleases Examples: Hind III: Eco RI: Pst I:
Objectives Before doing this lab you should understand how gel electrophoresis separates DNA molecules present in a mixture and how restriction endonucleases function. After doing this lab you should be
More informationBIOLOGY 163 LABORATORY. RESTRICTION MAPPING OF PLASMID DNA (Revised Fall 2017)
BIOLOGY 163 LABORATORY RESTRICTION MAPPING OF PLASMID DNA (Revised Fall 2017) Physical mapping of genomes is an important part of modern molecular genetics. As it's name implies, physical mapping seeks
More informationDNA Restriction Digestion Analysis
PR041 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DNA Restriction Digestion Analysis Teacher s Guidebook (Cat. # BE 307) think proteins!
More informationMolecular Techniques Third-year Biology
PLANNING Genetics Lab practices Molecular Techniques. Genetics Lab practices protocol. 2015-16 PCR-DIRECTED MUTAGENESIS, MOLECULAR CLONING AND RESTRICTION ANALYSIS Sessions 1 & 2 (2x3 hours): PCR-directed
More informationEZ-Vision DNA Dye as Loading Buffer
EZ-Vision DNA Dye as Loading Buffer Code Description Size N472-SAMPLE EZ-VIsion One, DNA Dye as Loading Buffer, 6X 0.3 ml N650-SAMPLE EZ-Vision Two, DNA Dye as Loading Buffer, 6X 0.3 ml N313-SAMPLE EZ-Vision
More informationLab 9 Restriction Enzyme Analysis
Name Assignment # Lab 9 Restriction Enzyme Analysis http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html 1) Define restriction enzyme 2) Define recognition sequence 3) Label the images
More informationDNA Laddering Assay Kit
DNA Laddering Assay Kit Item No. 660990 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 3 Precautions 4 If You
More informationRestriction Enzyme Analysis of DNA- Student Handout
Restriction Enzyme Analysis of DNA- Student Handout How to set up a restriction enzyme reaction Restriction enzymes (or restriction endonucleases) cleave DNA in a very specific fashion. Type II restriction
More informationImaging Nucleic Acid Gels on the Odyssey Fc Imager
Imaging Nucleic Acid Gels on the Odyssey Fc Imager Developed for: Odyssey Fc Imaging System Published September 2011. The most recent version of this protocol is posted at: http://biosupport.licor.com
More informationAGAROSE GEL ELECTROPHORESIS. Assiut University
AGAROSE GEL ELECTROPHORESIS By Prof. Dr. Asmaa Hussein Prof. of Zoonoses & Director of the MBRU Assiut University The standard method used to separate, identify electrophoresis and purify DNA fragments
More informationAGAROSE GEL ELECTROPHORESIS Modified from Wolbachia FIBR Project, Rochester University
AGAROSE GEL ELECTROPHORESIS Modified from Wolbachia FIBR Project, Rochester University Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular
More informationAnswer sheet. Student number:
Page 1 of 9 MIDTERM EXAM OF BIO/BPS3151 2016 Answer sheet Name: Student number: Part II: Calculations 1 128g 2 58.5g 3 NaCl: 1L Water: 0.2L 4 2.5 g/l 5 0.4 6 1:4:2 7 900 ml 8 Plasmid A: 3.75 µl Plasmid
More informationA Picofluor method for RNA quantitation using RiboGreen
A Picofluor method for RNA quantitation using 1. INTRODUCTION RNA Quantitation Reagent is an ultrasensitive fluorescent nucleic acid stain. It is a simple and rapid procedure for measuring RNA concentration
More information7.13 Experimental Microbial Genetics
MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms. 7.13 Fall 2008 Page
More informationELECTROPHORESIS 4 electrophoretic detection of DNA
ELITechGroup S.p.A. C.so Svizzera, 185 10149 Torino ITALY Offices: Tel. +39-011 976 19 Fax +39-011 936 76 11 E. mail: emd.support@elitechgroup.com WEB site: www.elitechgroup.com ASSAY PRINCIPLE The electrophoretic
More informationDetermination of Exclusion Effect in Wild Type and Rop Deficient Mutated pbr322 Co-transformations
Determination of Exclusion Effect in Wild Type and Rop Deficient Mutated pbr322 Co-transformations Suzana Sabaiduc and Andy Lo Microbiology and Immunology University of British Columbia pbr322 is an expression
More informationAnalysis of RNA by Analytical Polyacrylamide Gel Electrophoresis
CHAPTER SIXTEEN Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis Alexey Petrov, Albet Tsa, Joseph D. Puglisi 1 Stanford University School of Medicine, Stanford, CA, USA 1 Corresponding
More informationAbout us. Cyanagen s.r.l. has a certified Quality System ISO QUALITY CERTIFIED. Green Stain DNA Loading Dye Rev00
About us Cyanagen is a biotech company located in Bologna, dedicated to research, development and production of reagents for molecular diagnostic since 2003 and one of the leading companies in the field
More informationAgarose gel electrophoresis of DNA fragments
Agarose gel electrophoresis of DNA fragments Page 1 of 5 (Maniatis, Sambrook, BioWhittaker catalogue) Method: DNA in solution has a net negative charge due to its phosphate backbone (at the ph used during
More informationProduct Information FluoroStain DNA Fluorescent Staining Dye (Green, 10,000X)
www.smobio.com Product Information FluoroStain DNA Fluorescent Staining Dye (Green, 10,000X) DS1000 500 μl x 1 DS1001 500 μl x 5 Storage Protected from light 4 C for 12 months -20 C for 24 months Working
More informationGREEN STAIN DNA LOADING DYE DNA LOADING DYE CONTAINING FLUORESCENT STAIN FOR NUCLEIC ACID DETECTION IN GELS
GREEN STAIN DNA LOADING DYE DNA LOADING DYE CONTAINING FLUORESCENT STAIN FOR NUCLEIC ACID DETECTION IN GELS About us Cyanagen is a biotech company located in Bologna, dedicated to research, development
More informationProductInformation TECHNICAL BULLETIN MULTIPLE TISSUE NORTHERN BLOT, MOUSE. Product No. BLOT-2 Technical Bulletin No. MB-865 February 2000
MULTIPLE TISSUE NORTHERN BLOT, MOUSE Product No. BLOT-2 Technical Bulletin No. MB-865 February 2000 ProductInformation TECHNICAL BULLETIN Product Description Sigma s Mouse Multiple Tissue Northern Blot
More informationCONCEPTS AND METHODS INSTRUCTOR PLANNING AND PREPARATION
CONCEPTS AND METHODS This laboratory can help students understand several important concepts of modern biology: The relationship between genotype and phenotype. The use of transposable elements to mutagenize
More informationModifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning
Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning This experiment was designed by Dylan Dodd, based on research completed in Dr. Isaac Cann s lab*, with modifications and editing of content
More informationRapid DNA Ligation & Transformation Kit
1 Rapid DNA Ligation & Transformation Kit (#K1432 for 30 reactions) INTRODUCTION The Rapid DNA Ligation & Transformation Kit enables ligation of any type of DNA in 5 minutes at ambient temperature followed
More informationRFLP ANALYSIS OF DNA LABORATORY
RFLP ANALYSIS OF DNA LABORATORY BIG PICTURE You will be working with an essential research method widely used in genetics, conservation biology, and forensics. The lab is divided into three sections. Part
More informationA Comparison of the Effects of Microwave Irradiation and Heat Treatment of T4 and T7 Bacteriophage
A Comparison of the Effects of Microwave Irradiation and Heat Treatment of T4 and T7 Bacteriophage BONNIE BAINES Department of Microbiology and Immunology, UBC In an attempt to elucidate the mechanism
More informationTitle: Understanding the impact of orientation on gene expression of lux operon in pkn800 transformation into Escherichia coli DH5α
Seim - 1 Name: Darian Seim Title: Understanding the impact of orientation on gene expression of lux operon in pkn800 transformation into Escherichia coli DH5α Date: April 12 th, 2016 April 18 th, 2016
More informationA PCR Assay for the Anthocyaninless Mutation in Fast Plants and a Bridge Between Classical Genetics and Genomics
Tested Studies for Laboratory Teaching Proceedings of the Association for Biology Laboratory Education Volume 39, Article 61, 2018 A PCR Assay for the Anthocyaninless Mutation in Fast Plants and a Bridge
More informationLab 7: Running an Agarose Gel for the Restriction Digests
Lab 7: Running an Agarose Gel for the Restriction Digests A) Prepare a TAE agarose gel. (A more detailed protocol is in Lab 4) 1. Prepare 80 ml of 1% agarose gel in 1X TAE Gel buffer by heating in the
More informationRestriction Analysis of Purified para-r
Restriction Analysis of Purified para-r INTRODUCTION The restriction analysis will provide final proof that the cells transformed during Laboratory 6, cloned overnight in LB/amp and purified in Lab 10
More informationTable of contents. I. Description...2. Kit Components...2. Storage...2. Required reagents and equipment...2. V. Protocol...3. Example Experiment...
PCR FLT3/ITD Mutation Detection Set Cat.# 6632 Table of contents I. Description...2 II. III. IV. Kit Components...2 Storage...2 Required reagents and equipment...2 V. Protocol...3 VI. XII. Example Experiment...4
More informationAppendix IV Version
APPENDIX IV. Gel Electrophoresis. Migration of biological molecules in the presence of an electric field through a gel matrix is the heart of many biochemistry experiments. The variety of electrophoresis
More informationLab 2C: Basic Techniques. Dilute 10X TE Buffer to Make 1X TE Buffer Determine the Concentration of an Unknown DNA Sample Streak out bacteria colonies
Demonstration of sterile technique. Lab 2 Basic Techniques Lab 2A: Lab 2B: Lab 2C: Dilute 10X TE Buffer to Make 1X TE Buffer Determine the Concentration of an Unknown DNA Sample Streak out bacteria colonies
More informationGENERAL BIOLOGY LABORATORY II
Weeks 9-10: Bioassays of major biomolecules: Nucleic acids GENERAL BIOLOGY LABORATORY II Canbolat Gürses, Hongling Yuan, Samet Kocabay, Hikmet Geckil Department of Molecular Biology and Genetics Inonu
More informationPRODUCT INFORMATIOIN DNA blunting and Ligation
Product Code: BS243-BS244 PRODUCT INFORMATIOIN DNA blunting and Ligation Storage: Store at -20 o C. Avoid frequent thawing as this diminishes the quality of the kit. Description and Notes: 1. Construction
More informationGroup Members: Lab Station: BIOTECHNOLOGY: Gel Electrophoresis
BIOTECHNOLOGY: Gel Electrophoresis Group Members: Lab Station: Restriction Enzyme Analysis Standard: AP Big Idea #3, SB2 How can we use genetic information to identify and profile individuals? Lab Specific
More informationMERmaid Kit preps preps Filters/Catch Tubes Filters/Catch Tubes Filters/Catch Tubes
BIO 101 Protocol MERmaid Kit w eb With Optional SPIN Procedure Protocol Included Catalog Number Prep Size 1005-000 10 preps 1005-200 200 preps Optional 2080-400 40 Filters/Catch Tubes 2080-600 60 Filters/Catch
More informationMolecular Scissors: Lambda Digest Student Materials
Molecular Scissors: Lambda Digest Student Materials Introduction 2 Pre-Lab Questions. 5 Lab Protocol 6 Data Collection Worksheet. 9 Post-Lab Questions and Analysis.. 10 Plasmid Maps. 13 Last updated: August
More informationStudent Manual. Pre-Lab Introduction to DNA Fingerprinting STUDENT MANUAL BACKGROUND
BACKGROUND Pre-Lab Introduction to 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
More informationImaging Nucleic Acid Gels on the Odyssey Fc Imager
Imaging Nucleic Acid Gels on the Odyssey Fc Imager Developed for: Odyssey Fc Imaging System Published September 2011. The most recent version of this protocol is posted at: https://www.licor.com/bio/support/
More informationPART 1 LAB ESSENTIALS BACK TO LAB PROMOTION. Buy 2 Get 3rd Free. Get started in the lab with these great offers on lab essentials!
BACK TO LAB PROMOTION LAB ESSENTIALS PART 1 Get started in the lab with these great offers on lab essentials! DNA Markers and Protein Laddders DNA Polymerases and Agarose Buy 2 Get 3rd Free Plasmid Purification
More informationRestriction Enzymes and Lambda DNA
Restriction Enzymes and Lambda DNA Computer 6B Restriction enzymes have become an indispensable tool of molecular researchers over the past fifty years. This unique group of enzymes function as molecular
More informationFMF NIRCA PROTOCOL STEP 1.
FMF NIRCA PROTOCOL STEP 1. After you have isolated patient s DNA and DNA from a healthy donor (wild type), you perform a nested PCR. The primers used to amplify exon 2 and exon 10 of the mefv gene are
More informationCHAPTER 4A MAKING SURE YOU VE GOT A RECOMBINANT PLASMID. CHAPTER 4A STUDENT GUIDE 2013 Amgen Foundation. All rights reserved.
CHAPTER 4A MAKING SURE YOU VE GOT A RECOMBINANT PLASMID 55 INTRODUCTION When biologists clone a gene in order to produce human insulin, they create a recombinant plasmid that has the human insulin gene.
More information1. Why do DNA restriction fragments and plasmids separate when analyzed by gel electrophoresis?
INTRODUCTION When biologists clone a gene in order to produce human insulin, they create a recombinant plasmid that has the insulin gene. To do so, they use restriction enzymes to create DNA fragments
More informationDNA miniprep by Alkaline Lysis (activity)
DNA miniprep by Alkaline Lysis (activity) Contents 1 Alkaline Lysis 2 Exercise 1: Plasmid DNA Mini-Prep by Alkaline Lysis 3 Identification of Plasmid DNA 4 Exercise 2: Restriction Digestion Identification
More informationINDEX KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 BENEFITS
INDEX KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 BENEFITS ERRORE. IL SEGNALIBRO NON È DEFINITO. IMPORTANT NOTES 1 METHOD FOR DNA EXTRACTION FROM AGAROSE GELS 2 METHOD FOR DNA EXTRACTION FROM
More informationShort Tandam Repeat (D1S58) Detection Kit (for Academic Instructions) Product # 54600
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Short Tandam Repeat (D1S58) Detection Kit (for Academic Instructions)
More informationGel Electrophoresis and Analysis
6/28/2016 Gel Electrophoresis and Analysis B3 Summer Science Camp at Olympic High School Dr. Jennifer Weller Lab Method: Gel electrophoresis 2 6/28/2016 Electrophoresis: separating molecules in a charged
More informationPlasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions
Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions Maxim Biotech, Inc. 780 Dubuque Avenue, So. San Francisco, CA 94080, U.S.A. Tel: (800) 989-6296
More informationLab 5: Shark Attacks, Again! DNA Fingerprinting to the Rescue
Lab 5: Shark Attacks, Again! DNA Fingerprinting to the Rescue Notebook Lab Objectives Develop an understanding of the basic techniques used to study genetic polymorphisms encoded in DNA Gain familiarity
More informationEZ Vision DNA Dye as Loading Buffer
EZ Vision DNA Dye as Loading Buffer Code Description Size N472-1ML N472-KIT N650-1ML N650-KIT N313-1ML N313-KIT N473-2PK N473-3PK EZ Vision One DNA Dye as Loading Buffer, 6X EZ Vision Two, DNA Dye as Loading
More informationPlasmids. BIL 333 Lecture I. Plasmids. Useful Plasmids. Useful Plasmids. Useful Plasmids. ( Transfection ) v Small, circular, double-stranded DNA
BIL 333 Lecture I Plasmids v Small, circular, double-stranded DNA v Exogenous to genome! v Origin of Replication v Marker Gene v ( Reporter Gene ) Plasmids v Marker Gene Changes Phenotype Of Host v (Antibiotic
More informationExperiment 5. Restriction Enzyme Digest and Plasmid Mapping. VY NGUYEN 26 February 2016
Experiment 5 Restriction Enzyme Digest and Plasmid Mapping VY NGUYEN 26 February 2016 ABSTRACT 1. Understand the use of restriction enzymes as biotechnology tools 2. Become familiar with the principles
More informationPrepare CTAB solutions to extracting DNA from Plant
Prepare CTAB solutions to extracting DNA from Plant By Dr. Mona S. Alwahibi Botany and Microbiology Dep. Introduction The search for a more efficient means of extracting DNA of both higher quality and
More information1. Add 0.4 ml Phenol:Chloroform:Isoamyl alcohol and gently rotate tubes 3-4 hours at room temp. 2. Spin max. speed in microcentrifuge for 5 minutes.
High MW Genomic DNA Isolation Lysis Buffer: 100 mm NaCl 10 mm Tris ph 8.0 25 mm EDTA ph 8.0 0.5% SDS Lyse cells Tissue: Cut frozen tissues into small pieces with a razor blade, and grind into powder with
More informationReport on the Verification of Performance of a DNA Extraction Method for Maize Grains
Report on the Verification of Performance of a DNA Extraction Method for Maize Grains 7 September 2007 Joint Research Centre Institute for Health and Consumer Protection Biotechnology & GMOs Unit Method
More informationIntroduction. Kit components. 50 Preps GF-PC-050. Product Catalog No. 200 Preps GF-PC Preps GF-PC Preps SAMPLE
Introduction The GF-1 PCR Clean Up Kit is a system designed for rapid clean up of DNA bands ranging from 100bp to 20kb. The GF-1 PCR Clean Up Kit contains special buffers to provide the correct salt concentration
More informationDetection of Mutations by Single-strand Conformational
1 of 7 8/21/2008 10:22 AM Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3815 Protocol Detection of Mutations by Single-strand Conformational Polymorphism Joseph Sambrook and David W. Russell
More informationUse TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis.
1 of 6 4/29/2009 2:49 PM Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot3815 Protocol Detection of Mutations by Single-strand Conformational Polymorphism Joseph Sambrook and David W. Russell
More informationThe ramylase Project by Ellyn Daugherty
G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name The ramylase Project by Ellyn Daugherty Restriction Digestion to Verify pamylase Plasmid
More informationGel Electrophoresis: Quantitative length and mass measurements of DNA
BIO440 Genetics Lab Humboldt State University Gel Electrophoresis: Quantitative length and mass measurements of DNA Electrophoresis, and in particular agarose gel electrophoresis, is an integral analysis
More informationS.N.A.P. Gel Purification Kit
S.N.A.P. Gel Purification Kit For Rapid Purification of DNA Fragments from Agarose Gels Catalog nos. K1999-25 Version C 082701 25-0351 www.invitrogen.com tech_service@invitrogen.com ii Important Information
More informationAll-In-One Precast Agarose Gel Electrophoresis Kit (2x9-Well)
All-In-One Precast Agarose Gel Electrophoresis Kit (2x9-Well) Technical Manual No. 0282 Version 03242009 I Description... 1 II Key Features.. 1 III Safety Concerns... 2 IV Kit Contents.... 2 V Storage.....
More informationApplication of Molecular Biology tools for cloning of a foreign gene
IFM/Kemi Linköpings Universitet September 2013/LGM Labmanual Project course Application of Molecular Biology tools for cloning of a foreign gene Table of contents Introduction... 3 Amplification of a gene
More informationEXPERIMENT GENOMIC DNA ANALYSIS
EXPERIMENT GENOMIC DNA ANALYSIS Population diversity Studies We have 5 species of planarians (3 purchased from Carolina Biologicals, 2 obtained from the Levin lab) andmight have additional species found
More informationTexas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 8: DNA Restriction Digest (II) and DNA Sequencing (I)
Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 8: DNA Restriction Digest (II) and DNA Sequencing (I) We have made considerable progress in our analysis of the gene for
More informationCloneDirect Rapid Ligation Kit
CloneDirect Rapid Ligation Kit Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608) 831-9011 FAX: (608) 831-9012 lucigen@lucigen.com www.lucigen.com FOR RESEARCH
More informationThe Effect of Size of Chromosomal DNA from Escherichia coli VC10 on Transformation of Escherichia coli HB101 by the Plasmid p328.5
The Effect of Size of Chromosomal DNA from Escherichia coli VC10 on Transformation of Escherichia coli HB101 by the Plasmid p328.5 CHARLENE CHU, CHRISTINA HAN, HIROMI SHIMIZU, AND BONNIE WONG Department
More informationUser Manual. Topoisomerase I Assay Kit (plasmid based). Lot Number:
Copyright TopoGEN, Inc., 2012. All rights reserved. User Manual Topoisomerase I Assay Kit (plasmid based). Catalog Number Catalog Number TG1015-1 100 Reaction Set TG1015-2 250 Reaction Set Lot Number:
More informationPositive control kit for enzymatic mismatch cleavage and agarose gel visualization (version 2.4)
28 January, 2010 Positive control kit for enzymatic mismatch cleavage and agarose gel visualization (version 2.4) Plant Breeding Unit Brad Till and Owen Huynh January, 2010 Kit Contents: Genomic DNA from
More informationJustin Veazey. Experiment 3; Analysis of digestion products of puc19, GFPuv, and pgem-t easy
Veazey 1 Justin Veazey 7A Experiment 3; Analysis of digestion products of puc19, GFPuv, and pgem-t easy Construction of recombinants GFPuv-pGEM-T easy and GFPuv-pUC19 Transformation and analysis of recombinant
More informationLigation Independent Cloning (LIC) Procedure
Ligation Independent Cloning (LIC) Procedure Ligation Independent Cloning (LIC) LIC cloning allows insertion of DNA fragments without using restriction enzymes into specific vectors containing engineered
More information