Guide to Molecular Cloning Techniques

803 Pages·1987·13.406 MB·English

by  Abelson J.N., Berger S.L., Kimmel A.R. (eds.)

Preface
With the birth of recombinant DNA technology, the fields of nucleic
acid biochemistry and molecular biology entered an era marked by dra-
matic and innovative changes in methodology, rapid growth, and an al-
tered perception of how living systems could be studied. As additional
possibilities for applying these techniques were realized, it became evi-
dent that virtually every area of the life sciences would benefit. For the
specialist, this was a very exciting period, but for those who wished to
solve specific problems without first becoming full-time molecular biolo-
gists, the explosive increase in knowledge was overwhelming. Clearly,
the new technology had to be harnessed to serve biologists regardless of
scientific background.
This volume represents our contribution. Our aim is to meet the needs
of investigators entering molecular biology from other fields and to orient
students joining this discipline for the first time. We envisaged a self-
contained, concise compendium of state-of-the-art methods that might
also appeal to experienced individuals. To impose order on this complex
body of information, the book progresses from the basic techniques un-
derlying much of recombinant DNA technology to a series of sections,
each addressing a commonly met problem. Topics include genomic clon-
ing, preparation and characterization of mRNA, cDNA cloning, screening
libraries, and confirming the identity of selected clones.
The Guide contains reliable methods written by leaders in the field.
Many articles contrast different approaches for accomplishing the same
task in order to highlight strengths and weaknesses in a side-by-side com-
parison. Others present only the method that was deemed superior. To
assist the user, we have been assured that recommended vectors and
strains that are not commercially available will be provided by the au-
thors.
Because molecular cloning requires precise attention to detail, the
book is heavily edited in the form of cross referencing, Editors' Notes,
the Process Guide, and overviews. Pains have been taken to allow the
reader to pick and choose among articles without loss of continuity.
Cross-referencing helps to clarify the relationships among articles. So,
too, do the Editors' Notes. The Process Guide is another integrative
device in which fundamental processes in molecular biology are indexed
for ready accessibility. Since some methods are used frequently to
achieve not quite identical ends, the reader can locate at a glance those
that should be considered before choosing a specific technique. Finally,
XV
xvi ECAFERP
the volume contains overviews to five of the major sections to introduce
concepts and strategies and to aid in rapid recognition of relevant material
for a particular task.
Within the framework of a one-volume format, choices had to be
made. For example, Drosophila and Saccharomyces, organisms for
which specialized methods are abundant, have been short changed. Sepa-
rate volumes devoted exclusively to these subjects are required for ade-
quate coverage. Mutagenesis has not been emphasized but is covered
elsewhere in this series. Some attractive methods were not included be-
cause their utility and reproducibility are not firmly established. The
ediuG is primarily intended as an efficient means toward obtaining and
characterizing a clone. Within this narrow scope, the contents were cho-
sen based on what would be most important for most investigators most
of the time.
YBLEHS L. REGREB
ALAN R. KIMMEL
Contributors to Volume 152
era srebmun elcitrA ni sesehtnerap following the names of .srotubirtnoc
Affiliations listed era .tnerruc
DEBORAH A. ADAMS (8), Agouron The Insti- FRANK J. CALZONE (66), Division of Biol-
tute, 505 Coast Boulevard South, La ogy, California Institute of Technology,
Jolla, California 73029 Pasadena, California 52119
BARBARA J. B. AMBROSE (56), Chemical JOHN M. CrlIRGWIN (20), Division of Endo-
Abstracts Service, Columbus, Ohio crinology, Department of Medicine, ehT
01234 University of Texas Health Science Cen-
LYNNE M. ANGERER (68), Department of ter at San Antonio, San Antonio, Texas
Biology, University of Rochester, Roch- 48287
ester, New York 72641
FABIO COBIANCHI (10), lstituto di
ROBERT C. ANGERER (68), Department of Genetica Biochemica ed Evoluzionistica,
Biology, University of Rochester, Roch-
00172 Pavia, Italy
ester, New York 72641
CARL COSTANZI (62), Department of Neo-
WAYNE M. BARNES (57), Department of Bi-
plastic Diseases, Hahnemann University,
ological Chemistry, Washington Univer-
Philadelphia, Pennsylvania 20191
sity School of Medicine, St. Louis, Mis-
souri 01136 NANCY A. COSTLOW (67), Department of
Molecular Embryology, Memorial Sloan-
SHELBY L. BERGER (6, 19, 21, 26, 32, 33, 36,
Kettering Cancer Institute, New York,
37, 43, 45), Division of Cancer Biology
New York 12001
and Diagnosis, National Cancer Insti-
tute, National Institutes of Health, Be- KATHLEEN H. COX (68), Department of Bi-
thesda, Maryland 29802 ology, University of California at Los
Angeles, Los Angeles, California 42009
GRANT A. BITTER (70), Department of Mo-
lecular Biology, Amgen, Inc., 1900 Oak BRYAN R. ULLEN C (71), Department of Mo-
Terrace Lane, Thousand Oaks, California lecular Genetics, Hoffmann-La Roche
02319 Inc., Roche Research Center, Nutley,
New Jersey 01170
DAPHNE D. BLUMBERG (1, 2), Laboratory of
Comparative Carcinogenesis, National ERIC H. DAVIDSON (66), Division of Biol-
Cancer Institute, Frederick Cancer Re- ogy, California Institute of Technology,
Pasadena, California 52119
search Facility, Frederick, Maryland
10712 TTOCSERP L. DEININGER (41), Department
WILLIAM M. BONNER (7), Chromosome of Biochemistry and Molecular Biology,
Structure and Function Section, Labora- Louisiana State University Medical Cen-
tory of Molecular Pharmacology, Divi- ter, New Orleans, Louisiana 21107
sion of Cancer Treatment, National Can- ANTHONY G. DILELLA (18, 49), Depart-
cer Institute, National Institutes of ment of Cell Biology, Baylor College of
Health, Bethesda, Maryland 29802 Medicine, Houston, Texas 03077
RoY J. BRITTEN (66), Division of Biology, JAMES DOUGLAS ENGEL (40), Biochemistry,
California Institute of Technology, Pasa- Molecular Biology, and Cell Biology,
dena, California 52119 Northwestern University, Evanston, Illi-
JOAN E. BROOKS (11), New England nois 10206
BioLabs, Inc., Beverly, Massachusetts WILLIAM H. ESCHENFELDT (36, 37), Divi-
51910 sion of Cancer Biology and Diagnosis,
xi
xii CONTRIBUTORS TO VOLUME 152
National Cancer Institute, National Insti- (73), LOTHAR HENNIGHAUSEN Laboratory
tutes of Health, Bethesda, Maryland of Biochemistry and Metabolism, Na-
29802 tional Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes
GLEN A. (65), EVANS Cancer Biology Labo-
of Health, Bethesda, Maryland 29802
ratory, ehT Salk Institute, La Jolla, Cali-
fornia 83129 DRAHNREB G. NNAMRREH (15), Laboratory
of Molecular Embryology, National Insti-
YRAG (74), FELSENFELD Laboratory of Mo-
tute for Medical Research, Hill, Lon- Mill
Biology, lecular National Institute of Dia-
don NW7 1AA, England
betes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MICHAEL J. M. KCOCHCTIH (28, 29), Phar-
Maryland 29802 maceutical Research and Development
Division, Bristol-Myers Company, Wal-
JOHN C. (39), FIDDES Biotechnol- California
lingford, Connecticut 29460
ogy Inc., Bayshore 2450 Frontage Road,
Mountain View, California 34049 STEPHEN H. (50), HUGHES Frederick Can-
cer Research Facility, Frederick, Mary-
(15, FRISCHAUE ANNA-MARIA 16, 17), The
land 10712
Imperial Cancer Research Fund, Lin-
coln's Inn Fields, London WC2A 3PX, P. DAVID NOSKCAJ (74), Laboratory of Mo-
England Biology, lecular National Institute of Dia-
betes and Digestive and Kidney Diseases,
(62), GILLESPIE DAVID Department of Neo-
National Institutes of Health, Bethesda,
plastic Diseases, Hahnemann University,
Maryland 29802
Philadelphia, Pennsylvania 20191
ALLAN NOSBOCAJ (25), Department of Mo-
EDWARD I. GINNS (28), Molecular Neuro-
lecular Genetics and Microbiology, Uni-
genetics Unit, Clinical Neuroscience
versity of Massachusetts Medical School,
Branch, National Institute of Mental
Worcester, Massachusetts 50610
Health, National Institutes of Health, Be-
YRAMESOR (27, JAGUS 31, 60), Department
thesda, Maryland 29802
of Microbiology, Biochemistry and Mo-
NEHPETS P. GOFF (52), Department of Bio- lecular Biology, University of Pittsburgh
chemistry and Molecular Biophysics, -oC
School of Medicine, Pittsburgh, Pennsyl-
lumbia University, College of Physicians
vania 16251
and Surgeons, New York, New York,
JERRY JENDRISAK (40), Promega, 2800
23001
South Fish Hatchery Road, Madison,
NAHTANOJ R. GREENE (55), Department of Wisconsin 11735
Biology, Massachusetts Institute of Tech-
FOTIS C. (63, KAFATOS 64), Department of
nology, Cambridge, Massachusetts 93120
Cellular and Developmental Biology, The
DRANOEL (55), GUARENTE Department of Biological Laboratories, Harvard Univer-
Biology, Massachusetts Institute of Tech- sity, Cambridge, Massachusetts 02138,
nology, Cambridge, Massachusetts 93120 and Institute of Molecular Biology and
UELI (34, GUBLER 35), Department of Mo- Biotechnology and Department of Biol-
lecular Genetics, Hoffmann-La Roche, ogy, University of Crete, Heraclio, 71110
Inc., Nutley, New Jersey 01170 Crete, Greece
SALGUOD (39), HANAHAN Spring Cold Har- P. S. (44), KAYTES Molecular Biology Re-
bor Laboratory, Cold Spring Harbor, search, ehT Upjohn Company, Kalama-
New York 42711 zoo, Michigan 10094
DAVID M. HELFMAN (39, 50), Cold Spring ALAN R. (32, KIMMEL 42, 43, 54, 61), Labo-
Harbor Laboratory, Cold Spring Harbor, ratory of Cellular and Developmental Bi-
New York 42711 ology, National Institute of Diabetes and
CONTRIBUTORS TO VOLUME 152 xiii
Digestive and Kidney Diseases, National C. MIYADA GARRETT (47), Molecular Bio-
Institutes of Health, Bethesda, Maryland chemistry, Beckman Research Institute of
29802 the City of Hope, Duarte, California
CRAM S. KRUG (26, 33), Division of Cancer 01019
Biology and Diagnosis, National Cancer HPESOJ R. NEVINS (22), Laboratory of Mo-
Institute, National Institutes of Health, lecular Cell Biology, Howard Hughes
Bethesda, Maryland 29802 Medical Institute, The Rockefeller Uni-
SEMAJ J. LEE (67), Department of Genetics versity, New York, New York 12001
and Development, College of Physicians
DRAHCIR C. OGDEN (8), Agouron The Insti-
and Surgeons, Columbia University, New
tute, 505 Coast Boulevard South, La
York, New York 23001
Jolla, California 73029
HENRYK LUBON (73), Laboratory of Bio-
CHRIS PERCY (51), Promega, 2800 South
chemistry and Metabolism, National In-
Hatchery Fish Road, Madison, Wisconsin
stitute of Diabetes and Digestive and Kid-
11735
ney Diseases, National Institutes of
Health, Bethesda, Maryland 29802 PFEFFER DIANA (58, 59), Department of Bi-
ological Chemistry, University of -rofilaC
DENNIS C. LYNCH (24), Department of
ain at Los Angeles School of Medicine,
Medicine, Harvard Medical School,
Los Angeles, California 42009
Dana FaTher Cancer Institute, Boston,
Massachusetts 51120 ODLANYER C. PLESS (56), BRL/Life Tech-
nologies, Inc., Gaithersburg, Maryland
DNOMYAR J. MACDONALD (20), Depart-
77802
ment of Biochemistry, The University of
Texas Health Science Center at Dallas, ALAN E. PRZYBYLA (20), Department of
Dallas, Texas 53257 Biochemistry, University of Georgia, Ath-
LORAC J. ARUKES-SUCRAM (28, 29), Divi- ens, 2060Georgia 3
sion of Virology, Office of Biologics Re- TREBOR S. PUSKAS (37), Molecular Biology
search and Review, Food and Drug Ad- Department, Sigma Chemical Company,
ministration, Bethesda, Maryland 50202 St. Louis, Missouri 87136
DRANREB M. MECHLER (23), Institute of HTEBASILE A. RALEIGH (12), New England
Genetics, Johannes Gutenberg Univer- BioLabs, Inc., Beverly, Massachusetts
sity, D-6500 Mainz, Federal Republic of 51910
Germany
AHDARUNA YAR (38), Section of Biochemis-
JUDY L. MEINKOTH (9, 61), Gene Expres- try, Molecular and Cell Biology, Cornell
Laboratory, sion The Salk Institute for Bi- University, Ithaca, New York 35841
ological Studies, San Diego, California
KRAM D. ROSE (53), Department of Molecu-
83129
Biology, Princeton lar University, Prince-
SALGUOD A. MELTON (30), Department of ton, New Jersey 21580
Biochemistry and Molecular Biology,
MARTIN GREBNESOR (69), Biopharmaceuti-
Harvard University, Cambridge, Massa-
cal Research and Development, Smith
chusetts 83120
Kline and French Laboratories, 907 Swe-
TREBOR C. MIERENDORF (51, 58, 59), Pro- Avenue, deland Swedeland, Pennsylvania
mega, 2800 South Fish Hatchery Road, 97491
Madison, Wisconsin 11735
NADIA LAHTNESOR (72), Howard Hughes
YEVRAH MILLER (13), Department of Cell Medical Institute and Department of -raC
Genetics, Genentech, Inc., 064 Point San diology, The Children's Hospital, Har-
Bruno Boulevard, South San Franciso, vard Medical School, Boston, Massachu-
California 08049 setts 51120
xiv CONTRIBUTORS TO VOLUME 152
SAMOHT D. (46), SARGENT Laboratory of of Biology, Massachusetts Institute of
Molecular Genetics, National Institute of Technology, Cambridge, Massachusetts
Child Health and Human Development, 93120
National Institutes of Health, Bethesda, SAMUEL H. WILSON (10), Laboratory of
Maryland 29802 Biochemistry, National Cancer Institute,
ALLAN R. (69), SHATZMAN Biological Pro- National Institutes of Health, Bethesda,
cess Sciences Research and Develop- Maryland 29802
ment, Smith Kline and French Laborato- SAVlO L. C. Woo (18, 49), Howard Hughes
ries, 907 Swedeland Avenue, Swedeland, Medical Institute, Department of Bi- Cell
Pennsylvania 97491 ology, and Institute of Molecular Genet-
SUALOKIN A. LEREOPS (63, 64), Department ics, Baylor College of Medicine, Houston,
of Cellular and Developmental Biology, Texas 03077
The Biological Laboratories, Harvard WILLIAM I. WooD (48), Department of De-
University, Cambridge, Massachusetts velopmental Biology, Genentech, Inc.,
83120 064 Point San Bruno Boulevard, South
GALVIN H. SWIFT (20), Department of -GiB San Franciso, California 08049
chemistry, The University of Texas YAR WU (38), Section of Biochemistry, Mo-
Health Science Center at Dallas, Dallas, lecular and Biology, Cell Cornell Univer-
Texas 53257 sity, Ithaca, New York 35841
G. VOGELI (44), Molecular Biology Re- TIYUN WU (38), Section of Biochemistry,
search, The Upjohn Company, Kalama- Molecular and Biology, Cell Cornell Uni-
zoo, Michigan 10094 versity, Ithaca, New York 35841
GEOFFREY M. WAHL (9, 43, 45, 61, 65), ARLENE R. (14), WYMAN Department of Bi-
Gene Expression Laboratory, The Salk ology, Massachusetts Institute of Tech-
Institute, San Diego, California 83129 nology, Cambridge, Massachusetts 93120
DLANOD M. ECALLAW (4, 5), Genetics Unit, RICHARD A. YOUNG (40, 51), Whitehead
Glaxo Group Research Limited, Green- Institute for Biomedical Research and
ford, Middlesex 6BU 0HE, England Department of Biology, Massachusetts
R. BRUCE (47), WALLACE Molecular -GiB Institute of Technology, Cambridge,
chemistry, Beckman Research Institute of Massachusetts 93120
the City of Hope, Duarte, California ROBERT A. ZOON (3), Radiation Safety
01019 Branch, National Institutes of Health,
KENNETH F. (14), WERTMAN Department Bethesda, Maryland 29802
Process Guide
This listing contains the location by chapter number of specific methods
for which protocols have been provided. Generally, methods that are
mentioned but not presented in detail, or components of a reaction, are
not listed here. Thus, Filling in, Nick translation, and Primer extension
are in the Process Guide but DNA polymerase I and Klenow fragment
require consultation of the Subject Index.
A phenotype, testing for
Hfl mutation, 31
Absorbance, see also Nucleic acid detec- host restriction and modification
tion; Nucleic acid quantitation
systems, 31
of deoxyribonucleoside triphosphates, 6,
h lysogens, 31
47
P2 lysogens, 31
of double-stranded DNA, 6
recA mutation, 31
of oligonucleotides, 6, 47
suppressor mutations, 31
of RNA, 6
pure cultures, preparation of, 31
of single-stranded DNA, 6
storage of, 31
Actinomycin D, solution preparation of, 33
titering of, 31
Adaptors, use of, 38
Bentonite preparation, 2
Alkaline phosphatase, inhibition of, 61
Blots, see Nitrocellulose filters; Nylon
Annealing of tailed inserts to tailed vec-
membranes; and type of blot
tors, 37
Blots, dot
Antibody, see also Protein precipitation hybridization of, with labeled in vitro
for isolation of specific polysomes,
transcribed nuclear run on RNA,
preparation of, 24
22
primary, preparation of, 50, 15
immobilization of DNA on nitrocellulose
for screening h expression libraries,
for, 22, 62
preparation of, 15
immobilization of DNA on nitrocellulose
for screening plasmid expression li-
for, starting with intact cells, 62
braries, preparation of, 50
immobilization of RNA on nitrocellulose
secondary-alkaline phosphatase conju-
for, 62
gated, use of, 15
immobilization of RNA on nitrocellulose
secondary-t25I-labeled, use of, 50
for, starting with intact cells, 62
Autoradiography, 7
quantitation of in vitro transcribed nu-
of dried gels, 56, 74 clear run on RNA by means of,
of gels, 8 22
of sequencing gels, 56
Blots, hybridization, see Hybridization of
immobilized nucleic acids
Blots, Northem, 16
B
Blots, Southern, 16
Bacterial cultures, see also Competent for evaluation of long oligonucleotide
bacteria probes, 48
media, preparation of, 31 Blots-unblots, 47
xxxi
xxxii PROCESS GUIDE
C Chromosome walking, 46
cosmid vectors for, 56
CAT assay, 27 Colony hybridization, see also Hybridiza-
eDNA cloning tion of immobilized nucleic acids;
by addition of adaptors, 83 Libraries, cosmid and plasmid
by addition of linkers, ,83 93 master filters for, preparation of, ,81 44
directional, 93 master plates for, preparation of, ,81 44
of full-length molecules with vector- processing of replica filters for, ,81 44
primers, 14 purification of positive colonies, ,81 54
in 40 ~,gtl0, replica filters for, preparation of, ,81 44
in hgt ,11 40 Colony screening with antibodies, see also
overview, 23 Antibody; Libraries, plasmid
by sequential addition of different types master filters for, preparation of, 05
of linkers ,9 3 processing of replica filters for, 05
with tailed inserts and vectors, 73 purification of positive colonies, 05
eDNA, double stranded, purification of replica filters for, preparation of, 05
by G-75 Sephadex column chromatogra- Competenbta cteria
phy, 93 preparation of, 31
by Sepharose CL-4B column chromatog- transformation of, 31
raphy, ,63 93 Cytoplasm, isolation of, 12
eDNA, first strand, 33
as a hybridization probe, ,33 64
D
synthesis of, with methylmercury hy-
droxide, 33 DE-52 chromatography
synthesis of, with ,s1-2hTd(ogilo 33 for removal of triphosphates, 74
synthesis of, with random hexamers, 64 Deoxyribonuclease (DNase), treatment of
synthesis of, with specific primers, 66 to inhibit contaminating ribonuclease,
synthesis of, with vector-primers, ,33 12
14 Diazobenzyloxymethyi (DpBaMp)e r
eDNA, second strand immobilization of DNA on, 06
synthesis of, with hairpin-looped first Diazophenylthioether (DPT) paper
strand as primer, 43 immobilization of DNA on, 16
synthesis of, with mRNA fragments as Diethylpyrocarbonate treatment of solu-
primers, 53 tions, 2
synthesis of, with vector-primer con- DNA, blunt-ending of
structed eDNA, 14 with mung bean nuclease, ,53 47
cDNA, subtracted, 64 with T4 polymerase, ,01 53
Cell extract(eukaryotic) with ~S nuclease, ,43 66
preparation of, ,17 27 DNA, carrier, preparation of, 64
Cerenkov counting, 6 DNA, concentration (volume reduction)
Chloramphenicol acetyltransferase of
use of as a reporter gene, see CAT with butanol, 5, ,8 47
assay with a Centricon-30 device, 47
Chloramphenicol treatment DNA, cosmid, see also DNA, plasmid
for amplifying cosmids in bacteria on isolation of, small scale (miniprep), ,81
plates, 81 56
for amplifying plasmids inb acteria on DNA, digestion of, see also DNA probes;
plates, 44 Filling in; Nick translation; Phos-
for amplifying plasmids in bacteria in phates, 3'-terminal; Phosphates, 5'-
solution, 31 terminal
xxxiii
PROCESS GUIDE
bidirectionaUy with Bal 31, 01 DNA, M13
with Bal 31, single-strand-specific nucle- extraction of replicative form (RF), 31
ase activity of, 57 plus (+) strand, preparation of, 31
chemically for carrier, 46 preparation of, large scale, 31
by depurination in acid, 16 DNA, plasmid
with DNase I, ,01 73, 74 isolation of from Escherichia coli, small
with DNase I and ethidium bromide to scale (minipreps), ,31 58, 65
create nicks, 57 isolation of from Escherichia coli, large
with exonuclease III, ,01 57 scale, 31
with exonuclease VII, 66 isolation of from yeast, small scale, 35
internal single-stranded regions with Bal purification of
31, 57 by column chromatography, 31
with h exonuclease, 01 by CsCl-ethidium bromide equilibrium
into large random overlapping fragments gradient centrifugation, 31
with restriction enzymes, ,61 81 double cut, from excised small frag-
with mung bean nuclease, 35, 74 ment, 39
with restriction endonucleases, 11 DNA probes, preparation of, see also
with $I nuclease, 34, 39, 66 DNA sequencing, enzymatic methods
from 3'-termini with exonuclease III, 01 for; Filling in; Nick translation; Phos-
from 3'-termini with T4 polymerase, ,01 phates, 5'-terminal
35 asymmetric labeling of, 66, 74
from 5'-termini with h exonuclease, 01 cDNA, 33, 46
to widen nicks to gaps, 57 oligolabeling of (priming with random
DNA fractionation, see also DNA, ge- hexamers), 01
nomic, purification of; Electrophoresis oligonucleotides, 47, 48, 49
of nucleic acids oligonucleotides, tailed, 73
by hydroxylapatite chromatography to purification of, by gel electrophoresis, 66
separate double- from single- single-stranded from MI3 phage DNA
stranded molecules, 46, 74 templates, ,01 66, 74
in salt gradients, 81 single-stranded (partly) from M13 phage
in sucrose gradients, ,61 35 DNA templates, 01
DNA, genomic, preparation of DNA precipitation
from human blood, ,51 81 with ethanol, 5
from monolayer cultures of cells, ,51 81 with isopropanol, 5
from organs, 51 with spermine, 5
from yeast, 35 DNA sequencing
DNA, genomic, purification of, see also through base-paired regions (secondary
DNA fractionation; DNA, h, MI3, and structure), 58
plasmid by base-specific cleavage (Maxam-
in CsCI gradients, 35 Gilbert), 56
removal of RNA, 53, 74 by chemical modification, one procedure
DNA, h for four bases, 56
arms, preparation of, by enzymatic by dideoxynucleotidec hain termination,
digestion, 71 57, 58, 59
arms, purification of, in sucrose gradi- of denatured plasmid DNA, 58
ents, 71 enzymatic methods for, 57, 58, 59
isolation of, 31 use of dlTP to resolve compressions, 57
purification of of large fragments, 57
from kgtl0, 40 nested deletions for, 75
from hgtll, 40 of oligonucleotides, 56
xxxiv SSECORP GUIDE
polyacrylamide gels for, ,65 75 of polyacrylamide gels, 7, 13
of RNA transcripts synthesized in vitro, of tissues fixed in situ, 86
95 Footprinting in vitro, ,37 see also Protein
strategies for big projects, 75 binding in vitro
DNA sequencing data Footprinting in vivo, 47
acquisition of and handling, 75 use of solution hybridization for, 47
computer programs for, 75 use of Southern blots for, 47
Drop dialysis Formamide, deionization of, ,2 ,8 ,52 06
of nucleic acids, 5 suggestions for, 76
of proteins, 13 Fusion proteins
from hgtl I carrying lacZ
E production of, in lysogens, 15
purification of, suggestions, 15
Electrophoresis of nucleic acids, see also from pUC plasmids carrying lacZ
Gels
analysis of, 05
in agarose gels, denaturing
growth of bacteria for the production
with alkali, 8
of, 05
with formaldehyde, 8
from pAS plasmids
with glyoxai, 8
analysis of, 96
with methylmercury hydroxide, 8
Escherichia coli lysogenic host for, 96
in agarose gels, nondenaturing, 8
high-molecular-weight DNA in, ,61 81
mobility shift in the presence of protein, G
37
fl-Galactosidase, see also Fusion proteins;
neutralization of alkaline gels, 33
Isopropyl fl-D-thiogalactopyranoside
in polyacrylamide gels, denaturing
induction
with formamide, 8
use of as a reporter gene, 27
with urea, 8
Gels, see also Electrophoresis of nucleic
in polyacrylamide gels, nondenaturing, 8
acids
for purification, 8
autoradiography of, 7, 8, 65
of oligonucleotides from triphos-
dry, see also Blots-unblots
phates, 74
hybridization of, 74
of probes from 31M templates, 47
preparation of, ,7 ,8 ,33 47
for sequencoifn g DNA, ,65 75
elution of DNA from
for size determination, 8
by crush and soak, 55
Electrophoresis of proteins
with DE81 paper, 66
in the presence of DNA, 37
by diffusion, 8
in SDS polyacrylamide gels, ,13 96
by electroelution, 8, 55
Enhancer sequences, locationo f
by freeze-squeeze, 8
with reporter genes, 27
by melting low melting temperature
Evaporation, prevention of, ,33 76
agarose, ,8 55
Expression of cloned DNA
live 31M phage, 75
in Escherichia coli, ,05 ,15 96
sequencing of DNA in, ,65 75
in eukaryotic cells, ,07 17
Gene isolation
by complementation in yeast, 35
F
proof of, overview, 45
Filling in with Klenow fragment, ,01 ,93 75 by retroviral insertion, 25
Fluorography Genes, see also Genomic sequences;
of agarose gels, 7 Multigene families
of blots, 7 isolation of full-length, 46