Methods in Enzymology

Volume 152

Guide to Molecular CloningTechniques

Shelby L . Berger

Alan R . Kimmel

CONTRIBUTORS TO VOLUME 152 xi

PREFACE X V

VOLUMES IN SERIES XVii

PROCESS GUIDE XXXi

Section I . Requirements for a Molecular Biology Laboratory

1. Equipping the Laboratory

DAPHNE D . BLUMBERG

3

2. Creating a Ribonuclease-Free Environment

DAPHNE D . BLUMBERG

20

3 . Safety with J2 P- and 35 S-Labeled Compounds

ROBERT A . ZOON

25

Section II . General Methods for Isolating and Characterizing

Nucleic Acids

4. Large- and Small-Scale Phenol Extractions

DONALD M . WALLACE

33

5. Precipitation of Nucleic Acids

DONALD M . WALLACE

4 1

6 . Quantifying 32P-Labeled and Unlabeled Nucleic SHELBY L. BERGER

49

Acid s

7. Autoradiograms : 35S and 32P

WILLIAM M . BONNER

55

8 . Electrophoresis in Agarose and Acrylamide Gels RICHARD C . OGDEN AND

DEBORAH A . ADAMS

6 1

Section III . Enzymatic Techniques and Recombinant DNA Technology

9. Nick Translation

JUDY L . MEINKOTH AN D

GEOFFREY M . WAHL

9 1

10 . Enzymes for Modifying and Labeling DNA and FABIO COBIANCHI AN D

RNA

SAMUEL H . WILSON

94

Section IV. Restriction Enzyme s

11. Properties and Uses of Restriction Endonucleases JOAN E . BROOKS

11 3

12 . Restriction and Modification in Vivo by Escheri- ELISABETH A . RALEIGH

130chic coli K1 2

Section V. Growth and Maintenance of Bacteri a

13 . Practical Aspects of Preparing Phage and Plasmid HARVEY MILLER

14 5

DNA : Growth, Maintenance, and Storage of

Bacteria and Bacteriophage

Section VI . Genomic Cloning

14 . Host Strains That Alleviate Underrepresentation ARLENE R . WYMAN AN Dof Specific Sequences : Overview

KENNETH F . WERTMAN

173

15. Isolation of Genomic DNA

BERNHARD G . HERRMANN AN D

ANNA-MARIA FRISCHAUF

180

16. Digestion of DNA: Size Fractionation

ANNA-MARIA FRISCHAUF

183

17 . Construction and Characterization of a Genomic ANNA-MARIA FRISCHAUF

190Library in X

18 . Cloning Large Segments of Genomic DNA Using ANTHONY G . DILELLA AN DCosmid Vectors

SAVIO L . C . Woo

199

Section VII . Preparation and Characterization of RN A

19, Preparation and Characterization of RNA: Over- SHELBY L . BERGER

21 5view

20, Isolation of RNA Using Guanidinium Salts

RAYMOND J . MACDONALD ,

GALVIN H . SWIFT ,

ALAN E . PRZYBYLA, AN D

JOHN M . CHIRGWIN

21 9

21 . Isolation of Cytoplasmic RNA : Ribonucleoside- SHELBY L . BERGER

227

Vanadyl Complexe s

22. Isolation and Analysis of Nuclear RNA

JOSEPH R . NEVINS

23 4

23 . Isolation of Messenger RNA from Membrane- BERNARD M . MECHLER

24 1

Bound Polysomes

24. Use of Antibodies to Obtain Specific Polysomes DENNIS C . LYNCH

248

25. Purification and Fractionation of Poly(A) + RNA ALLAN JACOBSON

25 4

26 . Determination of the Molar Concentration ofMes- MARC S . KRUG AN D

senger RNA

SHELBY L . BERGER

262

27. Translation in Cell-Free Systems

ROSEMARY JAGUS

267

28 . Microinjection into Xenopus Oocytes : Equipment MICHAEL J . M . HITCHCOCK,

EDWARD I . GINNS, ANDCAROL J . MARCUS-SEKURA

276

29 . Preparation of Oocytes for Microinjection of RNA CAROL J . MARCUS-SEKURA ANDand DNA

MICHAEL J . M . HITCHCOCK 284

30 . Translation of Messenger RNA in Injected Frog DOUGLAS A . MELTON

28 8

Oocyte s

31. Characterization of in Vitro Translation Products ROSEMARY JAGUS

29 6

Section VIII . Preparation of cDNA and the Generation o f

cDNA Librarie s

32. Preparation of cDNA and the Generation of ALAN R . KIMMEL AND

cDNA Libraries : Overview

SHELBY L . BERGER

30 7

33 . First-Strand cDNA Synthesis Primed with MARC S . KRUG AN D

Oligo(dT)

SHELBY L . BERGER

31 6

34 . Second-Strand cDNA Synthesis : Classical UELI GUBLER

325

Metho d

35 . Second-Strand cDNA Synthesis : mRNA Frag- UELI GUBLER

33 0

ments as Primers

36 . Purification of Large Double-Stranded cDNA WILLIAM H . ESCHENFELD T

Fragments

AND SHELBY L . BERGER

33 5

37 . Homopolymeric Tailing

WILLIAM H . ESCHENFELDT ,ROBERT S . PUSKAS, AN DSHELBY L . BERGER

33 7

38. Adaptors, Linkers, and Methylation

RAY WU, TIYUN Wu, AN DANURADHA RAY

343

39 . Directional cDNA Cloning in Plasmid Vectors by DAVID M . HELFMAN ,

Sequential Addition of Oligonucleotide Linkers JOHN C . FIDDES, AN DDOUGLAS HANAHAN

349

40 . Cloning cDNA into XgtlO and Xgt11

JERRY JENDRISAK ,RICHARD A . YOUNG, AN DJAMES DOUGLAS ENGEL

359

41 . Full-Length cDNA Clones : Vector-Primed cDNA PRESCOTT L . DEININGER

37 1Synthesis

Section IX. Selection of Clones from Libraries

42. Selection of Clones from Libraries : Overview

ALAN R . KIMMEL

39 3

43 . Molecular Hybridization of Immobilized Nucleic GEOFFREY M . WAHL ,

Acids : Theoretical Concepts and Practical Con- SHELBY L . BERGER, AN D

siderations

ALAN R . KIMMEL

39 9

44 . Amplification, Storage, and Replication of Li- G . VOGELI AND

braries

P . S . KAYTES

40 7

45 . Screening Colonies or Plaques with Radioactive GEOFFREY M . WAHL AN D

Nucleic Acid Probes

SHELBY L . BERGER

41 5

46. Isolation of Differentially Expressed Genes

THOMAS D . SARGENT

42 3

47 . Oligonucleotide Probes for the Screening of Re- R . BRUCE WALLACE AN D

combinant DNA Libraries

C . GARRETT MIYADA

43 2

48 . Gene Cloning Based on Long Oligonucleotide WILLIAM I . WOOD

44 3Probe s

49 . Hybridization of Genomic DNA to Oligonu- ANTHONY G . DILELLA AN D

cleotide Probes in the Presence of Tetramethyl- SAN/lo L . C . Woo

44 7

ammonium Chlorid e

50 . Use of Antibodies to Screen cDNA Expression DAVID M . HELFMAN AN D

Libraries Prepared in Plasmid Vectors

STEPHEN H . HUGHES

45 1

51 . Gene Isolation by Screening 401 Libraries with ROBERT C . MIERENDORF,

Antibodies

CHRIS PERCY, AN D

RICHARD A. YOUNG

45 8

52. Gene Isolation by Retroviral Tagging

STEPHEN P . GOFF

46 9

53. Isolation of Genes by Complementation in Yeast MARK D . RosE

48 1

Section X . Identification and Characterization of Specific Clone s

54 . Identification and Characterization of Specific ALAN R . KIMMEL

50 7Clones : Strategy for Confirming the Validity o fPresumptive Clone s

55. Subcloning

JONATHAN R . GREENE AN D

LEONARD GUARENTE

512

56. DNA Sequencing : Chemical Methods

BARBARA J . B . AMBROS E

AND REYNALDO C . PLESS

522

57 . Sequencing DNA with Dideoxyribonucleotides as WAYNE M . BARNES

538Chain Terminators : Hints and Strategies for BigProjects

58. Direct Sequencing of Denatured Plasmid DNA

ROBERT C . MIERENDORF

AND DIANA PFEFFER

556

59 . Sequencing of RNA Transcripts Synthesized in ROBERT C . MIERENDORF

Vitro from Plasmids Containing Bacteriophage AND DIANA PFEFFER

56 3Promoters

60 . Hybrid Selection of mRNA and Hybrid Arrest of ROSEMARY JAGUS

567Translatio n

61. Northern and Southern Blots

GEOFFREY M . WAHL ,

JUDY L . MEINKOTH, AN D

ALAN R . KIMMEL

572

62 . Fast Blots : Immobilization of DNA and RNA CARL COSTANZI AN D

from Cells

DAVID GILLESPIE

58 2

63 . Identification of Genomic Sequences Correspond- NIKOLAUS A . SPOEREL AN D

ing to cDNA Clones

FOTIS C . KAFATOS

58 8

64 . Isolation of Full-Length Genes : Walking the Chro- NIKOLAUS A . SPOEREL AN D

mosome

FoTIS C . KAFATOS

59 8

65 . Cosmid Vectors for Genomic Walking and Rapid GLEN A . EVANS AN D

Restriction Mapping

GEOFFREY M . WAHL

60 4

66 . Mapping of Gene Transcripts by Nuclease Protec- FRANK J . CALZONE ,

tion Assays and cDNA Primer Extension

Roy J . BRITTEN, AN D

ERIC H . DAVIDSON

61 1

67 . A Molecular Titration Assay to Measure Tran- JAMES J . LEE AN D

script Prevalence Levels

NANCY A . COSTLOW

63 3

68 . Demonstration of Tissue-Specific Gene Expres- LYNNE M . ANGERER,

sion by in Situ Hybridization

KATHLEEN H. Cox, AN D

ROBERT C . ANGERER

64 9

69 . Expression, Identification, and Characterization ALLAN R . SHATZMAN AND

of Recombinant Gene Products in Escherichia MARTIN ROSENBERG

66 1col i

70. Heterologous Gene Expression in Yeast

GRANT A . BITTER

67 3

71 . Use of Eukaryotic Expression Technology in the BRYAN R . CULLEN

68 4Functional Analysis of Cloned Gene s

72 . Identification of Regulatory Elements of Cloned NADIA ROSENTHAL

70 4Genes with Functional Assays

73. Interaction of Protein with DNA in Vitro

LOTHAR HENNIGHAUSE N

AND HENRYK LUBON

724

74 . In Vivo Footprinting of Specific Protein-DNA In- P . DAVID JACKSON AN D

teractions

GARY FELSENFELD

73 5

AUTHOR INDEX 75 7

SUBJECT INDEX 783