Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-471 (Electronic) MOLECULAR BIOLOGY PROBLEM SOLVER
MOLECULAR BIOLOGY PROBLEM SOLVER Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
MOLECULAR BIOLOGY PROBLEM SOLVER A LABORATORY GUIDE Edited by Alan s. gerstein WWILEY-LISS A JOHN WILEY soNS INC. Publication New York· Chichester· Weinheim· Brisbane· Singapore· Toronto
MOLECULAR BIOLOGY PROBLEM SOLVER A LABORATORY GUIDE Edited by Alan S. Gerstein A JOHN WILEY & SONS, INC., PUBLICATION New York • Chichester • Weinheim • Brisbane • Singapore • Toronto
Designations used by companies to distinguish their products are often claimed as trademarks. In all instances where John Wiley Sons, Inc, is aware of a claim, product names appear in initial capital or ALL CAPITAL LETTERS. Readers, however, should contact the appropriate companies for more complete information regarding trademarks Copyright 2001 by Wiley-Liss, Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or ransmitted in any form or by any means, electronic or mechanical, including uploading downloading, printing, decompiling, recording or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without the prior written permission of the Publisher. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley Sons, Inc, 605 Third Avenue New York,NY10158-0012,(212)850-6011,fax(212)850-6008, EMail: PERMREOOWILEY. COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. ISBN0-471223905 This title is also available in print as ISBN 0-471-37972-7 For more information about Wiley products, visit our web site at
Designations used by companies to distinguish their products are often claimed as trademarks. In all instances where John Wiley & Sons, Inc., is aware of a claim, the product names appear in initial capital or all capital letters. Readers, however, should contact the appropriate companies for more complete information regarding trademarks and registration. Copyright © 2001 by Wiley-Liss, Inc. 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 or mechanical, including uploading, downloading, printing, decompiling, recording or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without the prior written permission of the Publisher. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ@WILEY.COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. ISBN 0-471-22390-5 This title is also available in print as ISBN 0-471-37972-7. For more information about Wiley products, visit our web site at www.Wiley.com
For Daniel and his mom
For Daniel and his Mom
Tis better to ask some of the questions than to know all of the answers Unknown Indiana
Tis better to ask some of the questions than to know all of the answers. Unknown, Indiana
Contents Preface Contributors Chapter I. Preparing for Success in the Laboratory Phillip P. Franciskovich Chapter 2. Getting What You Need from a Supplier Tom Tyre and greg Krueger Chapter 3. The Preparation of Buffers and other Solutions: A Chemists Perspective Edward A. Pfannkoch Chapter 4. How to Properly Use and Maintain Laboratory Trevor troutman Kristin a. Prasauckas. michele A Kennedy, Jane Stevens, Michael G. Davies, and Andrew T Dadd Chapter 5. Working Safely with Biological Samples Constantine G. Haidaris and Eartell ]. Brownlow Chapter 6. Working Safely with Radioactive Materials William R.. Volny ]r Chapter 7. DNA Purification 167 Chapter 8. RNA Purification ori A. Martin, Tiffany. Smith, Dawn Obermoeller Brian Bruner: Martin Kracklauer and Subramanian Dharmaraj
vii Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Chapter 1. Preparing for Success in the Laboratory Phillip P. Franciskovich . . . . . . . . . . . . . . . . . . . . . . . . 1 Chapter 2. Getting What You Need from a Supplier Tom Tyre and Greg Krueger . . . . . . . . . . . . . . . . . . . . 11 Chapter 3. The Preparation of Buffers and Other Solutions: A Chemist’s Perspective Edward A. Pfannkoch . . . . . . . . . . . . . . . . . . . . . . . . . 31 Chapter 4. How to Properly Use and Maintain Laboratory Equipment Trevor Troutman, Kristin A. Prasauckas, Michele A. Kennedy, Jane Stevens, Michael G. Davies, and Andrew T. Dadd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Chapter 5. Working Safely with Biological Samples Constantine G. Haidaris and Eartell J. Brownlow . . . . . 113 Chapter 6. Working Safely with Radioactive Materials William R. J. Volny Jr. . . . . . . . . . . . . . . . . . . . . . . . . . 141 Chapter 7. DNA Purification Sibylle Herzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Chapter 8. RNA Purification Lori A. Martin, Tiffany J. Smith, Dawn Obermoeller, Brian Bruner, Martin Kracklauer, and Subramanian Dharmaraj . . . . . . . . . . . . . . . . . . . . . . . 197 Contents
Chapter 9. Restriction Endonucleases Derek robinson paul r walsh and Joseph A. Bonventre Chapter 10. Nucleotides, oligonucleotides, and Polynucleotides Alan S Gerstein 267 Chapter II. PCR Kazuko Aoyagi 29l Chapter 12. Electrophoresis Martha L booz 33l Chapter 13. Western Blotting Peter Riis 373 Chapter 14. Nucleic Acid Hybridization Sibylle Herzer and David F Englert Chapter I5. E coli Expression Systems Peter A. Bell 46l Chapter 16. Eukaryotic Expression John Trill, Robert Kirkpatrick, Allan r. Shatzman, and Alice Marcy 491 Index 543
Chapter 9. Restriction Endonucleases Derek Robinson, Paul R. Walsh, and Joseph A. Bonventre . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Chapter 10. Nucleotides, Oligonucleotides, and Polynucleotides Alan S. Gerstein . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Chapter 11. PCR Kazuko Aoyagi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 Chapter 12. Electrophoresis Martha L. Booz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Chapter 13. Western Blotting Peter Riis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Chapter 14. Nucleic Acid Hybridization Sibylle Herzer and David F. Englert . . . . . . . . . . . . . . . 399 Chapter 15. E. coli Expression Systems Peter A. Bell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 Chapter 16. Eukaryotic Expression John J. Trill, Robert Kirkpatrick, Allan R. Shatzman, and Alice Marcy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543 viii Contents
Preface This book celebrates the importance of the question; it is not meant to be a collection of facts or procedures. The writing of this book was inspired by 16 years of queries from the research community. The con- tributors and i have tried to meet two primary objectives Enhance the reader's ability to identify the critical elements of any technique, reagent, or procedure, in order to address questions for which documented answers might be unavailable Clarify theory and practice that is taken for granted yet fre quently misapplied /hy is this book organized as a series of questions? For one, the esearchers(and people in general) who greatly impress me are those who when faced with a seemingly impassible dilemma, can identify the question(s) that point the way to an eventual solution. Second, I'm fairly certain that I was most useful to others when all I did was to help them identify the questions that enabled them to solve their own Who should read this book? I can only say that the contributing authors, many of whom work within a technical support group or have previously done so, were asked to compose their chapters based on uestions that they were chronically asked or based on questions that they wish had been asked by those requesting assistance. What are the strategies for working with this book? While Ive been arping on the importance of the question, you might only have time to locate an answer. For readers in search of quick information, you might want to begin your search with a review of the index. A second approach would be to review the tables of content at the beginning of each chapter, which list the questions addressed within them I strongly recommend that at some point you read through a chapter of interest, focusing only on the questions being asked and the sub headings contained in the answer. The authors and I would like to think that this information and the questions they inspire will provide insight and perspective to help you solve problems that go beyond the content of this book
This book celebrates the importance of the question; it is not meant to be a collection of facts or procedures. The writing of this book was inspired by 16 years of queries from the research community.The contributors and I have tried to meet two primary objectives: • Enhance the reader’s ability to identify the critical elements of any technique, reagent, or procedure, in order to address questions for which documented answers might be unavailable. • Clarify theory and practice that is taken for granted yet frequently misapplied. Why is this book organized as a series of questions? For one, the researchers (and people in general) who greatly impress me are those who when faced with a seemingly impassible dilemma, can identify the question(s) that point the way to an eventual solution. Second, I’m fairly certain that I was most useful to others when all I did was to help them identify the questions that enabled them to solve their own problems. Who should read this book? I can only say that the contributing authors, many of whom work within a technical support group or have previously done so, were asked to compose their chapters based on questions that they were chronically asked, or based on questions that they wish had been asked by those requesting assistance. What are the strategies for working with this book? While I’ve been harping on the importance of the question, you might only have time to locate an answer. For readers in search of quick information, you might want to begin your search with a review of the index. A second approach would be to review the tables of content at the beginning of each chapter, which list the questions addressed within them. I strongly recommend that at some point you read through a chapter of interest, focusing only on the questions being asked and the subheadings contained in the answer.The authors and I would like to think that this information and the questions they inspire will provide insight and perspective to help you solve problems that go beyond the content of this book. ix Preface
s So many friends, colleagues, and others who could have been clas- ied as competitors gave selflessly to make this project a reality. It is all too likely that someone will be forgotten, and to all those individ uals whose help I have not acknowledged, I sincerely apologize. Among those that I remember are Peter Herzer, Billi Herzer, N Booz, Alice Marcy, Bob Dunst, Mary Ann Fink, George Donzella, Kathie Gorski, Lou Hosta, Claire Wheeler John Graziadei, Joseph Stencel, Phil Franciskovich, Tom Myers, Holly Hogrefe, Carl Baker, John SantaLucia, Patti Taranto, Phil Beckett(Cheers, me old mucker), Howard Coyer, Anita Gradowski, David Remeta, Cica Minetti, Peter Chiang, Martha Cole, Matt Szap, Barb Aboard, Julie De Gregaro, and Paul Doderlein for the invaluable service they provided by reviewing manuscripts am grateful to Terri Sunquist and colleagues at Promega Biotech for data on RNa polymerases, Bengt Bjellavist for data on agarose, to Bjorn Lundgren for centrifugation data, to Bronwen Harvey and her research team for providing intriguing hybridization data, to Carl Fuller for sharing his contacts and enzyme expertise, and to Gene Stircak for access to search services. I am especially grateful to my colleague at Amersham Pharmacia Biotech for their support, good wishes and collective sense of humor. With such a talented group of supporters and contributing authors, you can blame me for any inadequacies you note within these pages There are probably innumerable people at john Wiley and Sons that I should include, but I can only name a few. Ann Boyle and virginia Benson Chanda have my sincere gratitude for their roles in convert- ing an idea into a publication. Special thanks go to my editor, Luna Han, for her bottomless well of patience and professional guidance I wish I could thank my parents, Bernard and Florence, who urged me to focus on learning, not test scores. I'm glad I can thank my wife Sharon for her love and ability to ignore my mood swings during this project, and my son Daniel, whose uncanny knack for getting me out of bed before dawn hastened me to the finish line Alan Gerstein P.S. Several authors (listed on page xi-xii) provide their electroni nail address to receive your inquiries and comments. I would greatly appreciate your forwarding me(mbproblemsolver@earthlink net )a copy of any correspondence you send to an author. Thank you in advance Preface
So many friends, colleagues, and others who could have been classified as competitors gave selflessly to make this project a reality. It is all too likely that someone will be forgotten, and to all those individuals whose help I have not acknowledged, I sincerely apologize. Among those that I remember are Peter Herzer, Billi Herzer, Martha Booz,Alice Marcy, Bob Dunst, Mary Ann Fink, George Donzella, Kathie Gorski, Lou Hosta, Claire Wheeler John Graziadei, Joseph Stencel, Phil Franciskovich, Tom Myers, Holly Hogrefe, Carl Baker, John SantaLucia, Patti Taranto, Phil Beckett (Cheers, me old mucker), Howard Coyer, Anita Gradowski, David Remeta, Cica Minetti, Peter Chiang, Martha Cole, Matt Szap, Barb Kaboard, Julie DeGregaro, and Paul Hoderlein for the invaluable service they provided by reviewing manuscripts. I am grateful to Terri Sunquist and colleagues at Promega Biotech for data on RNA polymerases, Bengt Bjellqvist for data on agarose, to Bjorn Lundgren for centrifugation data, to Bronwen Harvey and her research team for providing intriguing hybridization data, to Carl Fuller for sharing his contacts and enzyme expertise, and to Gene Stircak for access to search services. I am especially grateful to my colleagues at Amersham Pharmacia Biotech for their support, good wishes and collective sense of humor. With such a talented group of supporters and contributing authors, you can blame me for any inadequacies you note within these pages. There are probably innumerable people at John Wiley and Sons that I should include, but I can only name a few. Ann Boyle and Virginia Benson Chanda have my sincere gratitude for their roles in converting an idea into a publication. Special thanks go to my editor, Luna Han, for her bottomless well of patience and professional guidance. I wish I could thank my parents, Bernard and Florence, who urged me to focus on learning, not test scores. I’m glad I can thank my wife Sharon for her love and ability to ignore my mood swings during this project, and my son Daniel, whose uncanny knack for getting me out of bed before dawn hastened me to the finish line. Alan Gerstein P.S. Several authors (listed on page xi–xiii) provide their electronic mail address to receive your inquiries and comments. I would greatly appreciate your forwarding me (mbproblemsolver@earthlink.net) a copy of any correspondence you send to an author. Thank you in advance. x Preface
Contributors Aoyagi, Kazuko, Millennium Pharmaceuticals, Inc, Cambridge, MA Bell. Peter A, Orchid BioSciences Inc. Princeton Nj,pbell@orchid.com Bonventre, Joseph A, New England Biolabs, Inc., Beverly, MA, info@neb.com Booz. Martha L. Bio-Rad Laboratories Hercules CA martha boozobio-rad com Brownlow, Eartell ], University of Cincinnati College of Medicine, Cincinnati, OH Bruner. Brian. Ambion Inc. Austin TX Dadd, Andrew T, Biochrom, LTD, Cambridge, UK Davies, Michael G, Biochrom, LTD, Cambridge UK, enquiries @biochrom co uk Dharmaraj, Subramanian, Ambion Inc Austin TX Englert, David F, Packard Bioscience, Meriden, CT, support@packardinstrument.com Franciskovich, Phillip P, Motorola Life Sciences, Tempe AZ, apf008@email.mot.com
Aoyagi, Kazuko, Millennium Pharmaceuticals, Inc., Cambridge, MA Bell, Peter A., Orchid BioSciences, Inc., Princeton, NJ, pbell@orchid.com Bonventre, Joseph A., New England Biolabs, Inc., Beverly, MA, info@neb.com Booz, Martha L., Bio-Rad Laboratories, Hercules, CA, martha_booz@bio-rad.com Brownlow, Eartell J., University of Cincinnati College of Medicine, Cincinnati, OH Bruner, Brian, Ambion, Inc., Austin, TX Dadd,Andrew T., Biochrom, LTD., Cambridge, UK Davies, Michael G., Biochrom, LTD., Cambridge, UK, enquiries@biochrom.co.uk Dharmaraj, Subramanian, Ambion, Inc., Austin, TX Englert, David F., Packard Bioscience, Meriden, CT, support@packardinstrument.com Franciskovich, Phillip P., Motorola Life Sciences, Tempe AZ, apf008@email.mot.com xi Contributors
