Experiments in Molecular Biology

385 Pages·1986·16.11 MB·English

by  Elliot B. Gingold (auth.), Robert J. Slater (eds.)

Experiments in
Molecular Biology
Biological Methods
Experiments in Molecular Biology, edited by
Robert J. Slater, 1986
0/
Microcharacterization Polypeptides, edited
by John E. Shively, 1986
Methods in Molecular Biology, edited by John
M. Walker, 1984
Volume I: Proteins
Volume II: Nucleic Acids
Liquid Chromatography in Clinical Analysis,
edited by Pokar M. Kabra and Laurence J.
Marton, 1981
Metal Carcinogenesis Testing, by Max Costa,
1980
Experiments
•
In
Molecular Biology
Edited by
Robert J. Slater
Humana Press • Clifton, New Jersey
Library ofCongressCataloginginPublicationData
Main entryundertitle:
Experimentsinmolecularbiology
(Biologicalmethods)
Includesindex.
1. MolecularBiology-Experiments. I.Slater,
Robert J. II.Series
QH506.E96 1985 574.87'382 85-21882
ISBN0-89603- 082- 2
© 1986The HumanaPress Inc.
CrescentManor
PO Box2148
Clifton, NJ07015
Allrights reserved
Nopartofthisbookmaybereproduced,storedinaretrieval
system, ortransmittedinanyform orbyany means, electronic,
mechanical,photocopying,microfilming,recording,orotherwise
withoutwrittenpermissionfrom thePublisher.
Printedinthe UnitedStates ofAmerica
PREFACE
Research in the field of molecular biology has progressed at
a fascinating rate in recent years. Much of this progress
results from the development of new laboratory techniques
that allow very precise fractionation and analysis of nucleic
acids and proteins, as well as the construction of recom
binant DNA molecules that can then be cloned and
expressed in host cells. Progress has been so rapid that there
has been a shortfall in the training of appropriately qualified
staff. Many existing laboratory workers require retraining,
and many educational institutions have had difficulty incor
porating the new molecular biology techniques into their
teaching programs. Although there are several manuals
currently available that describe laboratory techniques in
molecularbiology, they are principallywritten for the indivi
dual research worker and are not intended for use in the
design of practical classes for students.
The aim of this book isto provide justsucha series of pro
tocols for the teaching of practical molecular biology. The
idea arose following the success of several Workshops in
Molecular Biology, organized and taught by staff in the Biol
ogy Department of the Hatfield Polytechnic. Gradually, the
protocols used in the workshops have been incorporated into
the Hatfield undergraduate and postgraduate teaching pro
grams and have now been collected together to form a book.
The lecturers and demonstrators in these workshops and
courses, together with respected authorities from other insti
tutions, have contributed their tried and tested protocols to
this book so that others may more readily incorporate
material central to modem molecular biology into their own
v
vi Preface
undergraduate and beginning graduate-level courses, or
indeed even develop similar laboratory workshops in their
own institutions.
The chapters following describe a range of the most
important techniques in molecular biology research today.
The book begins with chapters on the extraction and mani
pulation of DNA, continues with articles on RNA and pro
tein techniques, and closes with some experiments on whole
cells. In all but the first chapter, which is intended as an
introductory experiment, the procedures described are those
normally used in the research laboratory and have not been
simplified. This was done because the objectives here are to
teach participants in the experiments how the techniques are
routinely used and to provide a source book of procedures
that will be directly useful in their subsequent careers.
Nevertheless, the level of detail given is such that every
experimentshouldprove successfulat the first attempt.
Each chapter describes an experiment based on a particu
lar technique in molecular biology, and all are presented in
the same format. The Introduction gives a brief summary of
the techniques to be employed and describes the importance
of such techniques to current research. The aims and objec
tives of the experiment are then described and an indication
is given of the time required to complete the practical pro
cedures. The Materials Provided section lists all those items
that should be available before the practical work begins.
This is not intended to give all the details concerning the
equipment or buffers and solutions required, but is a check
list intended to help organization of the experiment and
ensurethatnothing has beenforgotten. The Protocol section
describes the practical procedure in detail, in numbered
chronologicalsteps. Whenan experimentinvolves a number
of separate stages, perhaps involving more than one session,
the Protocol is divided into discrete sections as appropriate.
The Results and Discussion section describes the predicted
results and gives details of any data manipulations that are
required. In many cases, interesting discussion points are
raised, or questions aboutdata interpretationare posed.
This book contains two Appendices. The first of these,
Appendix I, supplies general information relevant to many
of the preceeding chapters. Appendix II, on the other hand,
is divided into sections related to individual chapters and is
Preface vii
concerned with the preparatory work required to set up
each experiment. Appendix II is self-contained and
describes in detail any procedures that must be completed
prior to the practical class, lists the special equipment
required, and the amounts of materials, solutions, and
buffers needed for classes of 20 participants, who will be
assumed to work in pairs unless otherwise indicated. Some
experiments are best suited to smaller groups, whereas oth
ers can be expanded to teach much larger classes; this is
indicated where appropriate. It is assumed that basic
everyday laboratory equipment, such as pipets, bench
centrifuges, ice buckets, and waterbaths or incubators will
be available; these are often not listed.
Each chapter in the book has been written in a style suit
able for both teacher and student. If the reader is a teacher
of molecular biology, he or she should first select the
experiments of interest; the book can then be followed sys
tematically to form a whole course on the techniques of
molecular biology, or individual chapters can be chosen as
required. The Introduction will give an indication of the
time required to carry out the procedures, and Appendix II
can be given to the laboratory technician to prepare for the
practical class. The participants should first read the Intro
duction to become familiar with the experiment's purpose.
Then, before commencing the practical work, they should
read the Materials Provided section to be sure that all the
necessary items have been supplied. The experiment may
then proceed by following the steps described in the Proto
col. Finally, when the course has been completed, we hope
and expect that the participants will always feel able to
refer to the Protocol section and Appendix II as a resource
for future work in the field.
Robert J. Slater
Contents
Preface v
CHAPTER 1
DNAIsolation and Transformation in
Escherichia coli: An Introduction
ElliotB. Gingold
1. Introduction............................................................... 1
2. Materials Provided................................................... 3
2.1. Cultures 3
2.2. Solutions.......................................................... 3
2.3. Growth Media................................................. 3
2.4. Equipment 3
3. Protocol...................................................................... 3
3.1. DNA Isolation................................................. 4
3.2. Preparing the E. coli Recipient....................... 5
3.3. Transformation................................................ 5
4. Results and Discussion............................................ 6
CHAPTER2
Isolation of Plasmid DNA: The Cleared Lysate and
esCI Gradient Technique
Stephen A. Boffey
1. Introduction............................................................... 7
2. Materials Provided................................................... 10
2.1. Solutions and Chemicals 10
2.2. Equipment 10
ix
x
Contents
3. Protocol.............. 10
4. Results and Discussion 14
Reference.......................................................................... 15
CHAPTER3
Restriction Endonuclease Digestion and
Agarose Gel Electrophoresis ofDNA
StephenA. Boffey
1. Introduction............................................................... 17
2. Materials Provided................................................... 20
2.1. Solutions and Chemicals 20
2.2. Equipment 20
3. Protocol..... 20
3.1. Restriction of DNA 20
3.2. Casting of Agarose Gel..... 21
3.3. Loading and Running Gel............................. 23
4. Results and Discussion 24
References........................................................................ 27
CHAPTER4
Restriction Site Mapping
Alan Hall
1. Introduction 29
2. Materials Provided................................................... 32
2.1. Solutions 32
2.2. Agarose Gel Electrophoresis......................... 33
2.3. Apparatus and Equipment 33
3. Protocol......... 33
3.1. Day 1................................................................ 33
3.2. Day 2. 35
4. Results and Discussion 36
Reference.......................................................................... 38
Contents xi
CHAPTERS
Southern Blotting
AlanHall
1. Introduction............................................................... 39
1.1. Transfer Step 41
1.2. Preparation of Radioactive DNA.................. 41
1.3. Hybridization Step. 42
1.4. Washing Step.................................................. 43
2. Materials Provided................................................... 44
2.1. Electrophoresis................................................ 44
2.2. Southern Blotting...................................... 44
2.3. Hybridization and Washing.......................... 44
2.4. Nick Translation. 44
3. Protocol................................................................. 45
3.1. Day 1................................................................ 45
3.2. Day 2.................................................... 48
3.3. Day 3.. 49
3.4. Nick Translation Procedure..... 49
4. Results and Discussion 52
References 52
CHAPTER6
Preparative Electrophoresis: Extraction ofNucleic
Acids From Gels
Alan Hall
1. Introduction............................................................... 55
1.1. Extraction From Acrylamide Gels 55
1.2. Extraction From Agarose Gels.......... 56
2. Materials Provided... 58
2.1. Solutions for Digestion.................................. 58
2.2. Electrophoresis................................................ 58
2.3. Gel Extraction 58
3. Protocol........ 58
3.1. Day 1................................................................ 58
3.2. Day 2................................................................ 61
4. Results and Discussion.......... 62