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SHORT PROTOCOLSIN MOLECULARBIOLOGYFourth Edition

A Compendium of Methods from ;Current Protocols in Molecular Biology

EDITORIAL BOARD ' , ' , '

Frederick M. AusubelMassachusetts General Hospital & Harvard Medical School' ' ' •

Roger BrentThe Molecular Sciences Institute, Berkeley, California -..

Robert E. KingstonMassachusetts General Hospital & Harvard Medical School

David D. Moore . ,Baylor College of Medicine ;

. Technische Universitat DarmstadiJ.G. seidman FACHBEREICH 10 — BIOLOGIEHarvard Medical School — B i b I i o t h e k —

J m Schnittspahnstralie 10John A. Smith p f 4 2 8 7 D a r m s t a d tUniversity of Alabama at Birmingham " '"

„ . „ H inv.-Nr.Kevin StruhlHarvard Medical School

BB TU Darmstadt

Published by John Wiley & Sons, Inc. Ill III III II 111 I III52541778

New York • Chichester • Weinheim • Brisbane • Singapore • Toronto

CONTENTSxxiii Preface :-;xxvii Contributors

1 ESCHERICHIA COU, PLASMIDS, AND BACTERIOPHAGES

INTRODUCTION 1-1

1.1 Media Preparation and Bacteriological Tools 1-2Minimal Media - A-2Rich Media ..- ,• , ,,„ . ..-•, , 1 - 3Solid-Media , \- , . ',. , "" .', * ' 1-3T o p A g a r . , / -,".'.'""',.-. .. . 1-4S t a b A g a r . . . ' ' ' . ' . " ','.,','•- . "-.-. 1-4T o o l s ' . '''.. 1-4

1.2 G r o w t h i n L i q u i d M e d i a v>, 1_5Basic Protocol 1: Growing an Overnight Culture . •• •> ' . • • ' . :• •.. 1-5Basic Protocol 2: Growing Larger Cultures ; • 1-5Basic Protocol 3: Monitoring Growth .-. . 1-6

1.3 Growth on Solid Media 1-6Basic Protocol 1: Titering and Isolating.Bacterial'Colonies by Serial Dilutions 1-6Basic Protocol 2:-Isolating Single Colonies by Streaking a Plate . . ,, , 1 - 7Basic Protocol 3: Isolating Single Colonies by Spreading a Plate * 1-7Support Protocol 1: Replica Plating - - • 1-8Support Protocol 2f Strain Storage and Revival: • • ' ' . 1-8

• • ' • ' , 1 • - • • ; • ' - ' • ,_.-.

1.4 Selected Topics from Classical Bacterial Genetics . 1-9

1.5 MapsofPlasmids * 1-13C h o o s i n g a P l a s m i d V e c t o r . . . ." '-'-,','*"'/'• '•'. , ' . '".'.,'" 1 -15

1 .6 M i n i p r e p s o f P l a s m i d D N A ,, : . : . ; j ; . o . 1 - 2 2Basic Protocol 1: Alkaline Lysis Miniprep , • . - , , 1-22Alternate Protocol: Alkaline Lysis in 96-Well Microtiter Dishes . * ., . 1-22Basic Protocol 2: Boiling Miniprep , , .."_' ' , ' ^ c / . 1-23Support Protocol: Storage of Plasmid DNA " ' ' " ' * 1-24

1.7 Large-Scale Preparation of Plasmid DNA ' ' . ' , ' ' 1-24Preparation of Crude Lysates . ' _ . ,-r." ' ' . t . ^' r 1-24

Basic Protocol 1: Alkaline Lysis . ' s ' . * " ^ ' . ' 1-24Basic Protocol 2: CsCl/Ethi'dium Bromide Equilibrium Centrifugation' '" ' 1-25

Alternate Protocol: Plasmid DNAsPurification by Anion-Exchange or Size-Exclusion :

Chromatography ' ^ •> 1-26

1.8 Introduction of Plasmid DNA into Cells . ' . 1-27Basic Protocol 1: Transformation Using Calcium Chloride • •- j_27Alternate Protocol: One-Step Preparation and TransfofmationJof Competent Cells 1-28Basic Protocol 2: High-Efficiency Transformation by Electropbratidn . ' 1-29

1.9 Introduction to Lambda Phages , . , 1-30Lytic Growth • , 1-31Lysogenic Growth • ~> 1-32

1.10 Lambda as a Cloning Vector ^ 1-33Advantages of Using Lambda '*' ' • ' ' : 1-33Selections for Inserted DNA . - : • . . ^ . 1 - 3 3Maps of Lambda-Derived Cloning Vectors , . . . 1-36

1.11 Plating Lambda Phage to Generate Plaques - 1-36Basic Protocol 1: Isolating a Single Plaque by Titering Serial Dilutions " ' r 1-36Basic Protocol 2: Phage Transfection and In Vitro Packaging 1-37

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1.12 ~ Growing Lambda-Derived Vectors 1-38Basic Protocol: Making a Stock of Phage by Plate Lysis 1-38Alternate Protocol: Making a Liquid Lysate 1-38

1.13 Preparing Lambda DNA from Phage Lysates 1-39Basic Protocol: Preparing DNA by Step- and Equilibrium-Gradient Centrifugation ' ' 1-39 -

1.14 Introduction to Vectors Derived from Filamentous Phages 1-41-

1.15 Preparing and Using M13-Derived Vectors 1-44~*~ Basic Protocol: Preparing Single-Stranded DNA from Plasmids Using Helper Phage 1-44

2 PREPARATION AND ANALYSIS OF DNA

INTRODUCTION 2-1

2.1 A Purification and Concentration of DNA from Aqueous Solutions 2-3Basic Protocol: Phenol Extraction and Ethanol Precipitation of DNA ' ' 2-3Alternate Protocol 1: Precipitation of DNA Using Isopropanol 2-4Support Protocol 1: Buffering Phenol and Preparing Phenol/ Chloroform/Isoamyl Alchohol 2-4Support Protocol 2: Concentration of DNA Using Butanol 2-5Support Protocol 3: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction. 2-5Alternate Protocol 2: Purification of DNA Using Glass Beads 2-6Alternate Protocol 3: Purification and Concentration of RNA and Dilute Solutions of DNA 2-6Alternate Protocol 4: Removal of Low-Molecular-Weight Oligonucleotides and Triphosphates

by Ethanol Precipitation > . 2-7

2.1B Purification of DNA by Anion-Exchange Chromatography . 2-8

2.2 Preparation of Genomic DNA from Mammalian Tissue 2-9

2.3 Preparation of Genomic DNA from Plant Tissue 2-10Basic Protocol: Preparation of Plant DNA Using CsCl Centrifugation 2-10Alternate Protocol: Preparation of Plant DNA Using CTAB 2-11

2.4 Preparation of Genomic DNA from Bacteria 2-12Basic Protocol: Miniprep of Bacterial Genomic DNA ' 2-12Alternate Protocol: Large-Scale CsCl Prep of Bacterial Genomic DNA ' ' ' 2-13Support Protocol: Removal of Polysaccharides from Existing Genomic DNA Preps ' 2-14

2.5A Agarose Gel Electrophoresis | • • • • • - • ' 2-14Basic Protocol: Resolution ofLarge DNA Fragments on Agarose Gels' ' <J 2-14Support Protocol: Minigels and Midigels ., ' ' 2-16

, 2.5B Pulsed-Field Gel Electrophoresis . 2-16Basic Protocol: Field-Inversion Electrophoresis ' 2-16Alternate Protocol: Chef Electrophoresis , • ' 2-17Support Protocol: Preparation of High-Molecular-Weight DNA Samples and Size Markers 2-18

2.6 Isolation and Purification of Large DNA Restriction Fragmentsfrom Agarose Gels , 2-20

Basic Protocol 1: Electroelution from Agarose Gels ,. , 2-20Basic Protocol 2: Electrophoresis onto_NA-45 Paper " " . • ' ' . 2-21Basic Protocol 3: Isolation of DNA Fragments Using^Low

Gelling/Melting Temperature Agarose Gels ^ , 2-22Alternate Protocol 1: Recovery of DNA from Low Gelling/Melting

Temperature Agarose Gels Using (3-Agarase Digestion • '<- 2-23Alternate Protocol 2: Recovery of DNA from Low Gelling/Melting

Temperature Agarose Using Glass Beads 2-23Support Protocol: Rapid Estimation of DNA Concentration by

Ethidium Bromide Dot Quantitation . 2-24

2.7 Separation of Small DNA Fragments by Conventional Gel Electrophoresis 2-24Basic Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis 2-24Alternate Protocol: Electroelution of Small DNA Fragments from Polyacrylamide Gels 2-27Basic Protocol 2: Sieving Agarose Gel Electrophoresis 2-27

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2.8 Capillary Electrophoresis of DNA . , 2-28Instrumentation - . • • ; " : 2-28Separation Theory ; . • . • . . • • • . 2-29Strategic Planning , 2-29Basic Protocol 1: Separation of Oligonucleotides AV 2-31Basic Protocol 2: Quantitative PCR Analysis , 2-33Alternate Protocol: Genotyping . 2-34

2.9A Southern Blotting 2-34"" , Basic Protocol: Southern Blotting onto a Nylon or Nitrocellulose <* "• •

Membrane with High-Salt Buffer 2-34Support Protocol: Calibration of a UV Transilluminator * 2-37Alternate Protocol 1: Southern Blotting onto a Nylon Membrane

with an Alkaline Buffer . 2-37Alternate Protocol 2: Southern Blotting by Downward Capillary Transfer ' 2-38

• '- Alternate Protocol 3: Electroblotting from a PolyacrylamideGel to a Nylon Membrane • 2-38

2.9B Dot and Slot Blotting of DNA 2-39Basic Protocol: Dot and Slot Blotting of DNA onto Uncharged

Nylon and Nitrocellulose Membranes Using a Manifold ' ' 2-40Alternate Protocol 1: Dot and Slot Blotting of DNA onto a -..-..•

Positively Charged Nylon Membrane Using a Manifold 2-41Alternate Protocol 2: Manual Preparation of a DNA Dot Blot . . 2-41

2.10 Hybridization Analysis of DNA Blots • •,.., 2-42Basic Protocol: Hybridization Analysis of a DNA Blot with a

Radiolabeled DNA Probe ' 2-42Alternate Protocol: Hybridization Analysis of a DNA Blot with a

Radiolabeled RNA Probe 2-44Support Protocol: Removal of Probes from Hybridized Membranes 2-48

2.11 Purification of Oligonucleotides Using Denaturing Polyacrylamide .'., *;. /;Gel Electrophoresis . . - • 2-49

3 ENZYMATIC MANIPULATION OF DNA AND RNA

INTRODUCTION ' 3 - 1

3.1 Digestion of DNA with Restriction Endonucleases " 3-2Basic Protocol: Digesting a Single DNA Sample with a Single

Restriction Endonuclease ' : 3-2Alternate Protocol!: Digesting DNA with Multiple Restriction Endonucleases ' 3-3Alternate Protocol 2: Digesting Multiple Samples of DNA • 3-6Alternate Protocol 3: Partial Digestion of DNA with Restriction Endonucleases • -; 3-7Support Protocol: Methylation of DNA , u 3-7

3.2 Mapping by Multiple Endonuclease Digestions ' ' vi 3-8

3.3 Mapping by Partial Endonuclease Digestions 3-9

3.4 Reagents and Radioisotopes Used to Manipulate Nucleic Acids . 3-9Stock Solutions • • ••'•• ' 3 9

lOx Enzyme Buffers : .." J . • •• . 3 _ 1 0

Enzyme Reaction Conditions and Application's' . " : ' ' : ' r - ' " 3-13Nucleoside Triphosphates' • • ' r»1 i • 3-13

• Radioisotopes for Labeling Nucleic Acids ' ' : - • '» ' 3-16Basic Protocol: Measuring Radioactivity in DNA and RNA by Acid Precipitation ; 3-16Alternate Protocol: Spin-Column Procedure for Separating

Radioactively Labeled DNA from Unincorporated dNTP Precursors ' 3-17

3.5 DNA-Dependent DNA Polymerases 3-18Klenow Fragment of E. coli DNA Polymerase I 3-18

Basic Protocol 1: Labeling the 3' Ends of DNA 3-19

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Basic Protocol .2: Repairing 3' or 5' Overhanging Ends to GeneratBlunt Ends 3-20

Basic Protocol 3: Labeling of DNA by Random Oligonucleotide-Primed Synthesis 3-20

T4 DNA Polymerase • 3-21Native T7 DNA Polymerase 3-23Modified T7 DNA Polymerase 3-23Taq DNA Polymerase 3-24

3.6 Template-Independent DNA Polymerases •, . 3-25

3.7 RNA-Dependent DNA Polymerases 3-26 y

3.8 DNA-Dependent RNA Polymerases 3-27

3.9 DNA-Independent RNA Polymerases 3-28

3.10 Phosphatases and Kinases < 3-28Alkaline Phosphatases: BAP and CIP . 3-28T4 Polynucleotide Kinase . 3-29

Basic Protocol 1: Labeling 5' Ends by the Forward Reaction 3-29Basic Protocol 2: Labeling 5' Termini by the Exchange Reaction 3-29

3.11 Exonucleases 3-30Single-Stranded 5'->3' and 3'->5' Exonucleases 3-30Double-Stranded 5'->3'Exonucleases 3-30Double-Stranded 3'-»5' Exonucleases . 3-30

3.12 Endonucleases 3-31Bal 31 Nuclease 3-31SI Nuclease 3-32Mung Bean Nuclease ' 3-32Micrococcal Nuclease 3-33Deoxyribonuclease I (DNase I) , 3-33

3.13 Ribonucleases 3-34Ribonuclease A 3-34Ribonuclease H . . 3-34Ribonuclease Tl 3-35

3.14 DNA Ligases 3-36T4 DNA Ligase 3-36E. coli DNA Ligase 3-36

3.15 RNA Ligases . „. 3-36T4 RNA Ligase . • . 3-36

3.16 Subcloning of DNA Fragments ' 3-37Basic Protocol ' 3-37Alternate Protocol: Ligation of DNA Fragments in Gel Slices _ > 3-39

3.17 Constructing Recombinant DNA Molecules by the PolymeraseChain Reaction 3-39

Basic Protocol: Subcloning DNA Fragments . ^39

3.18 Labeling and Colorimetric Detection of Nonisotopic Probes 3-43Basic Protocol 1: Preparation of Biotinylated Probes by

Nick Translation ,. 3-43Basic Protocol 2: Preparation of Biotinylated Probes by Random Oligonucleotide-Primed Synthesis 3-45Support Protocol: Colorimetric Detection of Biotinylated Probes . 3-45Alternate Protocol: Preparation and Detection of Digoxigenin-

Labeled DNA Probes , , . 3-47


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3.19 Chemiluminescent Detection of Nonisotopic Probes , 3-47Basic Protocol: Chemiluminescent Detection of Biotinylated Probes 3-47Alternate Protocol: Chemiluminescent Detection of Digoxigenin-Labeled Probes ' "• J3-5OSupport Protocol: Calibrating an-Ultraviolet Light Source . ,3-50

4 PREPARATION AND ANALYSIS OF RNA

INTRODUCTION . ' . . , . ' 4-1

4.1 Preparation of Cytoplasmic RNA from Tissue Culture Cells ' 4-2Basic Protocol . . 4-2

. • Support Protocol: Removal of Contaminating DNA 4-3

4.2 Guanidinium Methods for Total RNA Preparation 4-4Basic Protocol: Single-Step RNA Isolatioivfrom Cultured Cells or Tissues 4-4

: Alternate Protocol'!: CsCl Purification of RNA from Cultured Cells 4-5Alternate Protocol 2: CsCl Purification of RNA from Tissue 4-6

4.3 Phenol/SDS Method for Plant RNA Preparation 4-7

. . 4.4 Preparation of Bacterial RNA . 4-8Basic Protocol 1: Isolation ofHigh-Quality RNA from Gram-Negative Bacteria 4-8Basic Protocol 2: Isolation of RNA from Gram-Positive,Bacteria 4-10Alternate Protocol: Rapid Isolation of RNA from Gram-Negative Bacteria 4-11

4.5 Preparation of Poly(A)+RNA 4-11• • - • • ? - • • . '

4.6 SI Analysis of Messenger RNA Using Single-Stranded DNA Probes 4-12Basic Protocol: SI Analysis of mRNA Using M13 Template . , 4-12Alternate Protocol 1: Synthesis of Single-Stranded Probe from . ' .

Double-Stranded Plasmid Template ... , 4-15Alternate Protocol 2: Quantitative SI Analysis of mRNA Using

Oligonucleotide Probes . . 4-15Support Protocol: Controls for Quantitative SI Analysis of mRNA ' 4-16

4.7 Ribonuclease Protection Assay • ' , * • . . , . • 4-17Basic Protocol _ r . . . 4-17Support Protocol 1: Gel Purification of RNA Probes , , , . 4-18Support Protocol 2: Preparation of Template DNA ' , 4-19

4.8 Primer Extension : . v i" . 4-19

4.9 Analysis of RNA by Northern and Slot Blot Hybridization r 4-21Basic Protocol: Northern Hybridization of RNA Fractionated by Agarose-Formaldehy'de Gel

Electrophoresis . . . .„, ' 4-22Alternate^^Prptocoll: Northern. Hybridization of RNA Denatured['by Glyoxal/DMSp Treatment 4-24Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Imrnobilized by Slot Blotting 4-25Support Protocol: Removal of Probes from Northern Blots. " ' ' * "'' 4-26

5 CONSTRUCTION OF RECOMBINANT DNA LIBRARIES

INTRODUCTION 5-1

5.1 Overview of Genomic DNA Libraries 5-2Representation and Randomness 5-3Subgenomic Libraries 5-3Vectors for Genomic DNA Libraries ' 5-3

5.2 Overview of cDNA Libraries """"' . ' 5-4

5.3 Amplification of a Bacteriophage Library 5-5

5.4 Amplification of Cosmid and Plasmid Libraries • ' ' ' 5-6


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6 SCREENING OF RECOMBINANT DNA LIBRARIES

INTRODUCTION . 6-1

' ' 6.1 Plating and Transferring Bacteriophage Libraries ' ' •' 6-3

6.2 Plating and Transferring Cosmid and Plasmid Libraries , 6-4

6.3 Using DNA Fragments as Probes 6-6Basic Protocol: Hybridization in Formamide ' •' -J 6-6

— Alternate Protocol: Hybridization in Aqueous Solution - . : • ;. -,•• i J . 6-6

6.4 Using Synthetic Oligonucleotides as Probes •' • 6-7Basic Protocol 1: Hybridization in Sodium Chloride/Sodium Citrate (SSC) : • • 6-7Basic Protocol 2: Hybridization in Tetramethylammonium Chloride (TMAC) v . ; , 6-8-Support Protocol: Labeling the 5' Ends of Mixed Oligonucleotides , ; . „ • • :; 6-10

6.5 Purification of Bacteriophage Clones . . " > . ! , g . j j• ' ' ... , -.0 :.**'".. ••'

6.6 Purification of Cosmid and Plasmid Clones 6-11

6.7 Immunoscreening of Fusion Proteins Produced in Lambda Plaques 6-12

Basic Protocol: Screening a X.gtl 1 Expression Library with Antibodies " ' 6-12Alternate Protocol: Induction of Fusion Proteiri'Expression with " . ' ;

IPTG Prior to Screening with' Antibodies : . 6-13

6.8 Immunoscreening after Hybrid Selection and Translation 6-14

6.9 Overview of Strategies for Screening YAC Libraries and* Analyzing YAC Clones " 6-16

Generat ing YAC Libraries ' ' ' " , ' ' . ''•-"" ,, 6-16YAC Library Screening by a Core Laboratory '"•'••'«' '• 6-16Designing a Locus-Specific P C R Assay for Screening ' . '" . 6-18Analyz ing Individual YAC Clones ' •''•'*" ' ' ' • • • • ' - 6 . 1 8

Construct ion and Analysis of a YAC-Insert Sublibrary , ; 6-196.10 Analysis of Isolated YAC Clones 6-20

Basic Protocol 1: Propagation and Storage of YAC-Containing"Yeast Strains '""„ ' 6-20; , , Basic Protocol 2: Preparation of YAC-Containing DNA from

Yeast Clones for Analysis by Southern Blotting . . ' 6-20Basic Protocol 3: Preparation of Yeast Chromosomes in Agarose ' - •• •

Plugs for Pulsed- Field Gel Electrophoresis 6-22Basic Protocol 4: End-Fragment Analysis Using;PCR Amplification . ... - 6-23Alternate Protocol: End-Fragment Analysis by Subcloning into a

Bacterial Plasmid Vector' ' " •••-•••• *' 6-26Support Protocol: Design and Preparation of pUC19-ES and pUC19-HS Subcloning Vector, 6-28Basic Protocol 5: Preparation of High-Molecular-Weight YAC-Containihg Yeast DNA in Solution 6-28Basic Protocol 6: Preparation and Analysis of a YAC-Insert Sublibrary , ." " -,' 6-30

7 DNA SEQUENCING

OVERVffiW OF DNA SEQUENCING METHODS 7-1

7.1 DNA Sequencing Strategies , s 7-8Dideoxy Sequencing . „, . , 7-8Chemical Sequencing ""' ' 7-9

7.2 Constructing Nested Deletions for Use in DNA Sequencing 7-11B asic Protocol 1: Using Exonuclease in to Construct Unidirectional Deletions -n 7-11Support Protocol 1: Protection of DNA from Exonuclease EH

Digestion Using [ot35S]dNTPs - ' "'• < 7-15Basic Protocol 2: Using Bal 31 Nuclease to Construct Nested Deletions • . 7-15Support Protocol 2: Preparation of M13mp Sequencing Vector DNA for Subcloning of

Bal 31-Digested DNA Fragments 7-20


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7.3 Preparation of Templates for DNA Sequencing • 7-21Basic Protocol 1: Preparation of Single-Stranded M13 Phage DNA 7-21Basic Protocol 2: Preparation of X DNA from Small-Scale Lysates ' '•'••• 7-22Basic Protocol 3: Miniprep of Double-Stranded Plasmid DNA •' - ),

for Dideoxy Sequencing . . . , ; * ! . 7-23Basic Protocol 4: Alkali Denaturation of Double-Stranded Plasmid

r DNA for Dideoxy Sequencing r 7-24Basic Protocol 5: Preparation of Plasmid DNA from an E.coli Colony

or Phage DNA from a Plaque for Thermal Cycle Sequencing . . . 7-25

7.4 DNA Sequencing by the Dideoxy Method •-.; • • !•- • 7-25Basic Protocol 1: Labeling/Termination Sequencing Reactions Using Sequenase (Modified T7

DNA Polymerase) \ . <,!. •' " '/: ' '" , ' * 7-26Alternate Protocol 1: Using Mn in the Labeling/Termination Reactions 7-30Alternate Protocol 2: Using Taq DNA Polymerase in the SangerProcedure '•"'•' 7-30Alternate Protocol 3: One-Step Sequencing Reactions Using 5'-End-Labeled Primers . , : 7-31Basic Protocol 2: Thermal Cycle Sequencing Reactions Using . . . - , . . •

cc-Labeled Nucleotides ,'• . ' 7-32Alternate Protocol 4: Thermal C v c ! e Sequencing Reactions.Using •. • , , • • -.<

5'-End-Labeled Primers -_• -;•• ', • , .,.. 7-33Alternate Protocol 5: Cycle Sequencing Using Fluorescence Dye-Labeled Primer (Dyeprimer)

or Terminator (Dyeterminator) , . . , 7-35

7.5 Dideoxy DNA Sequencing with Chemiluminescent Detection • 7-37Basic Protocol; DNA Sequencing Using Biotinylated Primers with Ghemiluminescent Detection 7-38Alternate Protocol 1: Two-Step (Indirect) Detection Using, . . . . . - •

Streptavidin and Biotinylated Alkaline Phosphatase s r .., 7-40Alternate Protocol 2: Sequencing with Hapten-Labeled Primers

and Detection with Antibody-Alkaline Phosphatase Conjugates * '''! 7-41

7.6 Denaturing Gel Electrophoresis for Sequencing . . . . 7-42Basic Protocol: Pouring, Running, and Processing Sequencing Gels '" .. ,: 7-43Alternate Protocol 1: Buffer-Gradient Sequencing Gels " ' ' " '' , ' : 7-46Alternate Protocol 2: Electrolyte-Gradient Sequencing Gels ' .•.'•. y _ ^Alternate Protocol 3: Formamide-Containing Sequencing Gels ~ ">'• ' •" '•'"•• ' M 7r47

7.7 Computer Manipulation of DNA and Protein Sequences ;*' , ' : " " . 7-47Sequence Data Entry •..<.>..£. 7 4 gSequence Data Verification and Assembly ' '. ' . ; ' d.'2 7-50Restriction Mapping v .. • , ' ^ ,;,/r:<i 7-52Prediction of Nucleic Acid Structure <:,/:. •••;!'. • 7-54Oligonucleotide Design Strategy ., i,-.» ' • 7-55Identification of Protein-Coding Regions _, . . • „ -. 7-57Homology Searching _.. . . . . . . ' . , -..;-./*" 7-58Genetic Sequence Databases and Other Electronic Resources ' '.'••• . ! . . , , !

Available to Molecular Biologists ' *'''. , __, ~* _"','. 7-60Appendix ' , ' < " ' . . ' ; 7-60

MUTAGENESIS OF CLONED DNA ^as - v

INTRODUCTION . . ', . s ; . ?t 8-1

8.1 Oligonucleotide-Directed Mutagenesis Without Phenotypic Selection' '''"''•' 's*' 8-2

8.2A Mutagenesis with Degenerate Oligonucleotides: Creating " ' ' '."Numerous Mutations in a Small DNA Sequence . , 8-5

8.2B Gene Synthesis: Assembly of Target Sequences Using Mutually . . : 'Priming Long Oligonucleotides ••• . • t. ; i ,! 8-8

8.3 Region-Specific Mutagenesis ';'"'[ 8-9Basic Protocol 8-9Support Protocol: Enrichment of Mutant Clones • 8-12


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8.4 Linker-Scanning Mutagenesis of DNA . 8-12Basic Protocol: Linker Scanning Using Nested Deletions and

Complementary Oligonucleotides • 8-12,Alternate Protocol: Linker Scanning Using Oligonucleotide-Directed Mutagenesis 8-14

8.5 Site-Directed Mutagenesis by the Polymerase Chain Reaction 8-15Basic Protocol 1: Introduction of Restriction Endonuclease

Sites by PCR . 8-15Basic Protocol 2: Introduction of Point Mutations by PCR 8-16

"'" • Alternate Protocol: Introduction of a Point Mutation bySequential PCR Steps . 8-21

9 INTRODUCTION OF DNA INTO MAMMALIAN CELLSOVERVIEW OF TRANSFECTION METHODS 9-1

i"-9.1 Calcium Phosphate Transfection • 9-6

Basic Protocol: Transfection Using Calcium Phosphate-DNAPrecipitate Formed in HEPES 9-6

Support Protocol 1: Glycerol/DMSO Shock of Mammalian Cells 9-7Alternate Protocol: High-Efficiency Transfection Using Calcium Phosphate-DNA Precipitate

Formed in BES , 9-8

9.2 Transfection Using DEAE-Dextran 9-9Basic Protocol: General Procedure for DEAE-Dextran Transfection 9-9

; Alternate Protocol 1: Experiment: Transfection to Test Enzyme Structure/Activity Relationships 9-11Support Protocol: Charcoal Stripping of Fetal Bovine Serum • 9-12

9.3 Transfection by Electroporation 9-13Basic Protocol: Electroporation into Mammalian Cells . 9-13Alternate Protocol: Electroporation into Plant Protoplasts 9-14

9.4 Liposome-Mediated Transfection . 9-15Basic Protocol: Transient Expression Using Liposomes , 9-15Alternate Protocol: Stable Transformation Using Liposomes 9-16

9.5 Selection of Transfected Mammalian Cells: Strategic Planning 9-16Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells . • . 9-19Basic. Protocol 2: Selectable Markers for Mammalian Cells 9-20

9.6 Overview of Genetic Reporter Systems ' :• .9-24Design of Reporter Vectors • 9-26In Vitro Reporter Assays • , .:,• , 9-26In Vivo Reporter Assays . • , • . r . 9-29

9.7A Isotopic Assay for Reporter Gene Activity : '' 9-33Basic Protocol 1: Chromatographic Assay for CAT Activity 9-33Alternate Protocol 1: In Situ Lysis of Cells for CAT Assay' ' ; ' 9-35Alternate Protocol 2: Phase-Extraction Assay for CAT Activity ' " ' 9-36Basic Protocol 2: Radioimmunoassay for Human Growth Hormone 9-37

9.7B Nonisotopic Assays for Reporter Gene Activity : • : : :;. 9-38Basic Protocol 1: Firefly Luciferase Reporter Gene Assay 9-38Alternate Protocol: Luciferase Assay in Freeze-Thaw-Lysed Cells - 9.39Basic Protocol 2: Chemiluminescent p-Galactosidase Reporter Gene Assay. . 9-40

9.8 Overview of the Retrovirus Transduction System 9-41Retrovirus Life Cycle 9-41Replication-Incompetent Vectors 9-44Replication-Competent Vectors . .. - 9^46Packaging Lines and Virus Production 9-46Murine Retroviruses , 9-50Avian Retroviruses " " 9-51Safety Issues , 9-52

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9.9 Preparation of a Specific Retrovirus Producer Cell Line •-,- 9-52Basic Protocol 1: Introduction of a Retrovirus Vector into a Packaging Cell Line , .;; 9-52Basic Protocol 2: Determination of Viral Titer: Identification of .

Producer Clones Making High-Titer Virus . " , . ' . . ' ' ' 9 -55Alternate Protocol:: Rapid Evaluation of Producer Colonies . ' 9-56Support Protocol: Xgal Staining of Cultured Cells 9-56

9.10 Transient Transfection Methods for Preparation of High-Titer .Retroviral Supernatants • • . • \ 9-57

Basic Protocol 1: Transient Transfection of a Retrovirus Vector into 293 Cells 9-57Support Protocol: Growth and Storage of 293 Cells r 9-58Basic Protocol 2: Infection of Adherent Cells with Retroviral Supernatant 9-59

. . Alternate Protocol 1: Infection of Adherent Cells by Spin Infection , 9-60Basic Protocol 3: Infection of Nonadherent Cells by Retroviral Supernatant 9-61Alternate Protocol 2: Infection of Nonadherent Cells by Cocultiyation •.. 9-62Alternate Protocol 3: Infection of Nonadherent Cells by Spin Infection 9-63

9.11 Large-Scale Preparation and Concentration of Retrovirus Stocks . 9-63Basic Protocol: Preparation of Virus Stock and Concentration by Centrifugation 9-64Alternate Protocol 1: Concentration by PEG Precipitation and Chromatography • 9-65Alternate Protocol 2: Concentration Using Molecular-Weight-Cutoff Filters 9-65

9.12 Detection of Helper Virus in Retrovirus Stocks ,. 9-66Basic Protocol: Detection of Helper Virus Through Horizontal Spread of Drug Resistance 9-66Alternate Protocol 1: Proviral Rescue to Detect Replication-Competent Retrovirus 9-67Alternate Protocol 2: Reverse Transcriptase Assay to Detect Helper Virus •'. :. -. 9-68

9.13 Retrovirus Infection of Cells In Vitro and In Vivo 9-69Infection of Cells In Vitro 9-69Infection of Rodents In Vivo 9-70

. ' . ' • ' ' ' • . i

10 ANALYSIS OF PROTEINS

OVERVIEW OF PROTEIN PURIFICATION AND CHARACTERIZATION 10-1

10.1 Determination of Protein Concentration 10-8Basic Protocol 1: Using A280 to Determine Protein Concentration . • .. . 10-8

• •' Basic Protocol 2: Using the Bradford Method to Determine Protein Concentration ' • 10-9

10.2 One-Dimensional SDS Gel Electrophoresis of Proteins 10-10Electricity and Electrophoresis ' 10-10Basic Protocol 1: Denaturing (SDS) Discontinuous Gel • ••' f

Electrophoresis: Laem'mli Gel Method . . 10-11Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems 10-16Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers 10-18Alternate Protocol 3: Continuous SDS-PAGE 10-19:Alternate Protocol 4: Casting and Running Ultrathin Gels 10-19Support Protocol 1: Casting Multiple Single-Concentration Gels 10-20Alternate Protocol 5: Separations of Proteins on Gradient Gels .. ; / .•."'. 10-22Support Protocol 2: Casting Multiple Gradient Gels : :.. 10-25Basic Protocol 2: Electrophoresis in Single-Concentration Minigels ,'..''. 10-27Support Protocol 3: Preparing Multiple Gradient Minigels » . , 10-29

10.3 Two-Dimensional Gel Electrophoresis Using the O'Farrell System 10-30Basic Protocol 1: First-Dimension (Isoelectric-Focusing) Gels1 ' ' 10-30Basic Protocol 2: Second-Dimension Gels 10-32Isoelectric Focusing of Very Basic and Very Acidic Proteins ' 10-34Alternate Protocol: Two-Dimensional Minigels - - 10-35Support Protocol: Solubilization and Preparation of Proteins in Tissue Samples J 10-35

10.4 Staining Proteins in Gels 10-36Basic Protocol 1: Coomassie Blue Staining 10-36Basic Protocol 2: Silver Staining 10-36Alternate Protocol: Nonammoniacal Silver Staining 10-37


CONTENTS Page xi

Basic Protocol 3: Rapid Silver Staining • 10-38Support Protocol: Gel Photography •: 10-39

10.5 Immunoblotting and Immunodetection 10-40Basic Protocol 1: Protein Blotting with Tank Transfer Systems 10-40Alternate Protocol 1: Protein Blotting with Semidry Systems . 10-42Alternate Protocol 2: Blotting of Stained Gels 10-43Support Protocol 1: Reversible Staining of Transferred Proteins <• 10-44Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody 10-44Alternate Protocol 3: Immunoprobing with Avidin-Biotin Coupling

to the Secondary Antibody 10-45Basic Protocol 3: Visualization with Chromogenic Substrates 10-46Alternate Protocol 4: Visualization with Luminescent Substrates 10-46Support Protocol 2: Stripping and Reusing Membranes 10-48

10.6 Gel-Filtration Chromatography 10-48Basic Protocol 1: Desalting (Group Separation) 10-48Basic Protocol 2: Protein Fractionation .10-54Basic Protocol 3: Determination of Molecular Size 10-58Support Protocol: Column Calibration . 10-60

10.7 Ion-Exchange Chromatography . 10-62Strategic Planning ' • .-.10-62Basic Protocol 1: Batch Adsorption and Step-Gradient Elution

with Increasing Salt Concentration 10-63Basic Protocol.2: Column Chromatography with Linear Gradient Elution 10-66Support Protocol 1: Test Tube Pilot Experiment to Determine Starting '

Conditions for Ion-Exchange Chromatography 10-68

10.8 Immunoaffinity Chromatography 10-73Basic Protocol: Isolation of Soluble or Membrane-Bound Antigens 10-73Alternate Protocol 1: Batch Purification of Antigens 10-76Alternate Protocol 2: Low-pH Elution of Antigens 10-76

10.9 Metal-Chelate Affinity Chromatography 10-76Basic Protocol: Native MCAC for Purification of Soluble • s f

Histidine-Tail Fusion Proteins •: • .: 10-77Alternate Protocol 1: Denaturing MCAC for Purification of Insoluble Histidine-Tail Fusion Proteins 10-79Alternate Protocol 2: Solid-Phase Renaturation of MCAC-Purifie Proteins „ ,10-81Support Protocol: NTA Resin Regeneration . 10-81

10.10 Reversed-Phase Isolation of Peptides i , 10-82Basic Protocol 1: Reversed-Phase Peptide Separation at the 5- to 500-pmol Level •,. 10-82Basic Protocol 2: Reversed-Phase Peptide Separation at <5 pmol ' 10-83Support Protocol: Capillary HPLC System Assembly 10-84

10.11 Purification of Recombinant Proteins by Epitope Tagging 10-86Basic Protocol 1: Immunoprecipitation of Epitope-Tagged Recombinant Proteins , 10-86

10.12 Immunoprecipitation 10-88Basic Protocol: Immunoprecipitation of Radiolabeled Antigen with Antibody-Sepharose : 10-88Support Protocol: Preparation of Antibody-Sepharose 10-90Alternate Protocol 1: Immunoprecipitation of Radiolabeled Antigen

with Anti-Ig Serum 10-91Alternate Protocol 2: Immunoprecipitation of Radiolabeled Antigen with Anti-Ig-Sepharose,

Protein A - or G-Sepharose, or 5. aureus Cells ' 10-92Alternate Protocol 3: Immunoprecipitation Using More Strongly

Dissociating Lysis and Wash Buffers ' 10-93Alternate Protocol 4: Immunoprecipitation of Unlabeled Antigen

with Antibody-Sepharose " 10-93

10.13 Synthesizing Proteins In Vitro by Transcription andTranslation of Cloned Genes 10-94


Pagexii ix CONTENTS

10.14 Metabolic Labeling with Amino Acids 10-96Safety Precautions for Working with'•> S-Labeled Compounds - - 10-96Basic Protocol: Pulse-Labeling of Cells in Suspension with [ S]Methionine 10-96Alternate Protocol 1: Pulse-Labeling of Adherent Cells with [S]Methionine 10-97Alternate Protocol 2: Pulse-Chase Labeling of Cells with I^5S]Methiohine 10-98Alternate Protocol 3: Long-Term Labeling of Cells with [ S]Methionine 10-99Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids ' ' , ; v *•' ••:'-. 10-100Support Protocol: TCA Precipitation to Determine Label Incorporation 10-101

10.15 Isolation of Proteins for Microsequence Analysis ' 10-102Basic Protocol 1: Determination of Amino Acid Sequence of Samples on • ' '-*

PVDF Membranes 10-102Support Protocol: Preparation of Protein Samples for SDS-PAGE . •>•••• 10-104Basic Protocol 2: Determination of Internal Amino Acid Sequence from . •

N-Terminally Blocked Proteins r :!• . - 10-104

11 IMMUNOLOGY

INTRODUCTION ' 11-1

11.1 Conjugation of Enzymes to Antibodies • • . . • 11-3Basic Protocol: Conjugation of HRPO to Antibodies . , . 11-3Alternate Protocol: Conjugation of Alkaline Phosphatase to Antibodies 11-4

11.2 Enzyme-Linked Immuriosorbent Assay (ELISA) , . 11-4Basic Protocol: Indirect ELISA to Detect Specific Antibodies ,:-. • 11-4Alternate Protocol 1: Direct Competitive ELISA to Detect Soluble

Antigens 11-6Alternate Protocol 2: Antibody-Sandwich ELISA to Detect Soluble

Antigens " ' . 11-8Alternate Protocol 3: Double Antibody7Sandwich ELISA to Detect,

Specific Antibodies - • . ^ ^Alternate Protocol 4: Direct Cellular ELISA to Detect Cell-Surface - ' r

A n t i g e n s < --i'fC '••>' :- ' '•"••'-• • ' ? ' ' : ; 1 1 - 1 1Alternate Protocol 5: Indirect Cellular.EMS A to Detect Antibodies

Specific for Surface Antigens - ;: I''•< '•'• '•"' ' . 11-12Support Protocol 1: Criss-Cross Serial Dilution Analysis to Determine -,'

Optimal Reagent Concentrations . 11-13Support Protocol 2: Preparation of Bacterial-Cell-Lysate Antigens ,! 11-14

11.3 Productionof Monoclonal Antibody Supernatant and'Ascites Fluids . ' . . : . , . , 11-15Basic Protocol 1: Production of a Monoclonal Antibody. Supernatant. , ."" . , 11-16Alternate Protocol, 1: Large-Scale Production of Monoclonal Antibody Supernatant 11-16Alternate Protocol 2: Large-Scale Production of Hybridomas or Cell Lines .,' 11-17Basic Protocol 2: Production of Ascites Fluid Containing Monoclonal Antibody . ' 11-18

^ ' • • . • . . . ' * • •

11.4 Purification of Monoclonal Antibodies . . 11-20Basic Protocol: Purification Using Protein A-Sepharose r ,: 11-20Alternate Protocol 1: Alternatiy,e Buffer System for Protein ,., _,

A-Sepharose . _ 11-20Alternate Protocol 2: Purification by AntigenrSepharose and Anti-mouse-Ig-Sepharose 11-21

11.5 Production of Polyclohal Antisera in Rabbits 11-22Basic Protocol: Intramuscular Immunization ^ 11-22Alternate Protocol 1: Intradermal Immunization * : ' ; 11-24Alternate Protocol 2: Subcutaneous Immunization ^ :'-"' • c . ' 11-24Alternate Protocol 3: Bleeding from the EapArtery •"... , 11-24

11.6 Purification of IgG.Antibodies from Antiserum," .' ".-Ascites Fluid, or Hybridoma Supernatant^, , ; . . ..,'. ' ',,, ., ,,•- ' 11-25

^ Basic Protocol: Precipitation ofjIgG.with Ammonium Sulfate . , • . , .., 11-25Alternate Protocol: Fractionation of IgG by Chromatography on

DEAE-Affi-Gel Blue 11-26

11.7 Selection of an Immunogenic Peptide 11-26

.___ . Short Protocols in Molecular Biology

CONTENTS Page xiii

. 11.8 Production of Antipeptide Antibodies • 11-28Basic Protocol: Chemical Coupling of Synthetic Peptide to

Carrier Protein Using MBS . . . . 11-29Alternate Protocol: Chemical Coupling of Synthetic Peptide to , -•„

Carrier Protein Using Glutaraldehyde . . . - , - , 11-29

12 DNA-PROTEIN INTERACTIONS

I N T R O D U C T I O N ' _ 1 2 - 1

1 2 . 1 P r e p a r a t i o n o f N u c l e a r a n d C y t o p l a s m i c E x t r a c t s f r o m _ . . , _ . . •M a m m a l i a n C e l l s , ,.-*.,• •• 1 2 - 3

Basic Protocol: Preparation of Nuclear Extracts •*;" ' 12-3Support Protocol 1: Optimization of Nuclear Extraction ... . '-,-. 12-5Support Protocol 2: Preparation of the Cytoplasmic (S-100) Fraction 12-6

12.2 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis 12-6Basic Protocol: Mobility Shift Assay ••• 12-7';

Alternate Protocol 1: Competition Mobility Shift Assay ,12-9'Alternate Protocol 2: Antibody Supershift Assay 12-10Alternate Protocol 3: Multicorriponent Mobility Shift Assays '.*• v •». 12-10

12.3 Methylation and Uracil interference Assays for Analysis of ;Protein-DNA Interactions 12-11

Basic Protocol!: Methylation Interference Assay • ' • . . . 12-11Basic Protocol 2: Uracil Interference Assay 12-13

12.4 DNase I Footprint Analysis of Protein-DNA Binding 12-15Basic Protocol: DNase I Footprint Titration .. 12-15Support Protocol: Quantitation of Protein-Binding Equilibria by

Densitometric arid Numerical Analyses . 12-18Alternate Protocol: DNase Footprinting in Crude Fractions 12-20

12.5 UVCrosslinking of Proteins to Nucleic Acids , 12-21Basic Protocol: UV Crosslinking Using a BrdU-Substituted Probe 12-21Alternate Protocol 1: UVCrosslinking Using a Non-BrdU-Substituted Probe 12-24Alternate Protocol 2: UV Crosslinking In Situ .. - 12-24

12.6 Purification of DNA-Binding Proteins Using Biotin/StreptavidinAffinity Systems " . 12-25

Basic Protocol '• • ' '" ' ' ' 12-25Alternate Protocol 1: Purification Using a Microcolumn " 12-27Alternate Protocol 2: Purification Using Streptavidin-A^arose 12-28

12.7 Detection, Purification, and Characterization of cDNA Clones 'Encoding DNA-Binding Proteins , / 12-28

Basic Protocol: Screening a Xgtl 1 Expression Library withRecognition-Site DNA ' r 12-28

Alternate Protocol: Denaturation/Renaturation Cycling of Dried Filters' • '' ' • ; 12-30Support Protocol: Preparation of a Crude Extract from a Recombinant Lysogen to Characterize

DNA-Binding Activity 12-31

12.8 Analysis of DNA-Protein interactions Using Proteins SynthesizedIn Vitro from Cloned Genes 1. U-32

12.9 Purification of Sequence-Specific DNA-Binding Proteins by • • .Affinity Chromatography . . ,.'•_- 12-33

Basic Protocol 1: Preparation of DNA Affinity Resin , , , , , . . ;,/ . , 12-33Alternate Protocol: Coupling the DNA to Commercially Available CNBr-Activated Sepharose 12-36Support Protocol 1: Purification of Oligonucleotides by Preparative GelElectrophoresis 12-41Basic Protocol 2: DNA Affinity Chromatography ' ' '• 12-38Support Protocol 2: Selection and Preparation of Nonspecific Competitor DNA 12-40


Page xiv CONTENTS

12.10 Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection ; ,12-41Basic Protocol : ?, - . : . . \ " j i i - 12-41Support Protocol: Isolation and Analysis of Bound Oligonucleotides . . ••? ,I rnss;'.

from Mobility Shift Gels ... ,, • . . . , , „ . . <;, 12-45

13 SACCHAROMYCES CEREYISIAE

INTRODUCTION

13.1

13.2

13.3

13.4

13.8

13.9

13.10

Preparation of Yeast MediaLiquid MediaSolid MediaStrain Storage and Revival

Basic Protocol: Preparation and Inoculation of Frozen Stocks,

Growth and Manipulation of YeastBasic Protocol 1: Growth in Liquid MediaBasic Protocol 2: Growth on Solid MediaBasic Protocol 3: Determination of Cell DensityBasic Protocol 4: Determination of Phenotype by Replica PlatingBasic Protocol 5: Diploid ConstructionBasic Protocol 6: Sporulation of Diploid CellsBasic Protocol 7: Preparation and Dissection of TetradsSupport Protocol: Preparation of Dissecting NeedlesAlternate Protocol: Random Spore Analysis * .

'•'ft 1:

Mutagenesis of Yeast CellsBasic Protocol: Mutagenesis Using Ethyl Methanesulfonate .•;: ,..Alternate Protocol: Mutagenesis Using UV Irradiation

'I : i .-..

Yeast Cloning Vectors and Genes , ,. • ., ;. . : . >•Plasmid Nomenclature „ \(,::{-:Maps of Selected Plasmids and Genes : .•-. ,c

13.5 Yeast Vectors for Expression of Cloned Genes -..' • «:;•". '

13.6 Yeast Vectors and Expression Assays • . " .Basic Protocol 1: LacZ Fusion Vectors "for Studying Gene Regulation '•"Basic Protocol 2:Assay for P-galactosidase in Liquid CulturesAlternate Protocol: Screening for p-Galactosidase-Expressing Yeast Colonies Using a '

Filter Lift Assay

13.7 Introduction of DNA into Yeast Cells 'Basic Protocol: Transformation Using Lithium Acetate ' • ' % I 1- ' ,Alternate Protocol 1: SpheroplastTransformation , . ' - • •• - "' "^Alternate Protocol 2: Transformation by'Electroporation ;Support Protocol: Preparation of Single-Stranded High-Molecular-Weight Carrier-DNA

Cloning Yeast Genes by Complementation * *'4 ' *!"v

, i ; u ? 3

I'd,-."."

S e g r e g a t i o n o f P l a s m i d s f r o m Y e a s t C e l l s ; • - , - • •• . " ; • •.<• • • • • - - - . } ' . \Basic Protocol 1 . . . •.>!/.

Basic Protocol 2: Plasmid Gap Repair , ; ,,? .. . i-' . • . .,IJABasic Protocol 3: Plasmid Shuffling

Manipulation of Cloned Yeast DNA , •.'-• .,.-u, ,-., -;;-,t(Basic Protocol 1: Integrative Transformation ., ,.!• otGene Replacement Techniques •• • • c "• /

Basic Protocol 2: Integrative Disruption . . : iiBasic Protocol 3: One-Step Gene Disruption :- '•, • -;jAlternate Protocol 1: PCR-Mediated One-Step Gene Disruption • iv , . i iBasic Protocol 4: Transplacement . ' •, . • ., • -.)»/•»-.. -,-•

Basic Protocol 5: Creating Modified Genes by One-Step Integrative Replacement SJ,;, ,t, AAlternate Protocol 2: Creating Modified Genes by Transplacement : , ,..rjfl o.;, isju1

Basic Protocol 6: Creation of Conditional Alleles by Copper-Inducible DoubleEShutoff Procedure

13-1

13-213-313-513-713-7

13-713-813-813-813-913-913-913-1013-1213-13

13-1413-14

.13-16

13-1613-17

.13-21

13-26

13-2813-2813-29

13-30

.13-3113-3113-3213-3413-35

13-36

13-3813-3813-3913-39

13-4113-4113-4213-4213-4213-4313-4413-4513-4713-47


CONTENTS Page xv

413.11 Preparation of Yeast DNA 13-49• ••' Basic Protocol: Rapid Isolation of Plasmid DNA from Yeast 13-49

Alternate Protocol: Rapid Isolation of Yeast Chromosomal DNA : 13-50

13.12 Preparation of Yeast RNA 13-51, Basic Protocol: Preparation of Yeast RNA by Extraction with Hot

Acidic Phenol - ' 13-51"Alternate Protocol 1: Preparation of RNA Using Glass Beads , 13-52Alternate Protocol 2: Preparation of Poly(A)+ RNA 13-53

13.13 Preparation of Protein Extracts from Yeast 13-53Basic Protocol: Spheroplast Preparation and Lysis 13-53Support Protocol: Nuclei Preparation by Differential Centrifugation 13-55Alternate Protocol 1: Cell Disruption Using Glass Beads 13-55Alternate Protocol 2: Cell Disruption Using Liquid Nitrogen 13-56

14 IN SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY

INTRODUCTION 14-1

14.1 Fixation, Embedding, and Sectioning of Tissues, Embryos, andSingle Cells 14-2

Basic Protocol: Paraformaldehyde Fixation and Paraffin WaxEmbedding of Tissues and Embryos 14-2

Alternate Protocol: PFA Fixation of Suspended and Cultured Cells 14-3Support Protocol 1: Perfusion of Adult Mice 14-4Support Protocol 2: Sectioning Samples in Wax Blocks . . . -14-5Support Protocol 3: Preparation of Coated Slides . -.- 14-6

14.2 Cryosectioning ' 14-7Basic Protocol: Specimen Preparation and Sectioning 14-7Support Protocol 1: Fixation of Cryosections for In Situ Hybridization - • ' 14-9Support Protocol 2: Tissue Fixation and Sucrose Infusion ' ' 'JOi. • 14-11

14.3 In Situ Hybridization to Cellular RNA : . 14-11Basic Protocol: Hybridization Using Paraffin Sections or Cells . * . - . . . 14-11Alternate Protocol: Hybridization Using Cryosections « ' • •)•' 14-15Support Protocol 1: Synthesis of S-Labeled Riboprobes , ,. 14-16Support Protocol 2: Synthesis of S-Labeled Double-Stranded

DNA Probes : • ' , • • • • • • ' .. ' ' ' 14-17

14.4 Detection of Hybridized Probe 14-17Basic Protocol 1: Film Autoradiography .. 14-18Basic Protocol 2: Emulsion Autoradiography ' ' 14-18

, Support Protocol: Preparation of Diluted Emulsion for Autoradiography ' 14-19

14.5 Counterstaining and Mounting of Autoradiographed In SituHybridization Slides 14-19

Basic Protocol: Giemsa Staining 14-19'Alternate Protocol 1: Hematoxylin/Eosin Staining •': 14-21

Alternate Protocol 2: Toluidine Blue Staining • 14-21Alternate Protocol 3: Hoechst Staining 14-22

14.6 Immunohistochemistry 14-22Basic Protocol 1: Immunofluorescent Labeling of Cells Grown as

Monolayers 14-23Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells 14-23Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections 14-24Alternate Protocol 2: Immunofluorescent Labeling Using Streptavidin-

Biotin Conjugates • >" 14-25Alternate Protocol 3: Immunogold Labeling of Tissue Sections 14-26Alternate Protocol 4: Immunoperoxidase Labeling of Tissue Sections 14-26Alternate Protocol 5: Immunofluorescent Double-Labeling of Tissue

-•Sections • ' . . ' 14-28r f {. -


Pagexvi CONTENTS

14.7 In Situ Hybridization and Detection Using Nonisotopic Probes 14-2?Basic Protocol: Fluorescence In Situ Hybridization . 14-29Amplification of Hybridization Signals .14-31

Support Protocol 1: Amplification of Biotinylated Signals 14-31Support Protocol 2: Amplification of Signals from Digoxigenin-

Labeled Probes' , ' S ' ' 14-32Enzymatic Detection of Nonisotopically Labeled Probes 14-33

Alternate Protocol 1: Enzymatic Detection Using Horseradish Peroxidase 14-33Alternate Protocol 2: Enzymatic Detection Using Alkaline Phosphatase 14-34

14.8 In Situ Polymerase Chain Reaction and Hybridization to DetectLow-Abundance Nucleic Acids Targets ' 14-35

Strategic Planning " " 14-35Basic Protocol 1: In Situ PCR (ISPCR) Amplification of DNA and . • • ' •:.

RNA Targets with In Situ Reverse Transcription for RNA •: " ' • 14-38Alternate Protocol: One-Step Reverse Transcription and Amplification 14-42Basic Protocol 2: Hybridization and Detection of ISPCR-Amplified • ,

Target Material . » 14-43Support Protocol 1: Preparation .of AES-Subbed Slides . . 14-45Support Protocol 2: Preparation of Specimens on Slides.for ISPCR . 14-45Support Protocol 3: Labeling Oligosaccharide Probes Using T* ,' 14-46

15 THE POLYMERASE CHAIN REACTION

INTRODUCTION 15-1

15.1 Enzymatic Amplification of DNA by PCR: Standard Proceduresand Optimization . , . 15-3

15.2 Direct DNA Sequencing of PCR Products . 15-8Basic Protocol 1: Dideoxy" Sequencing of Single-Stranded Products Generated by Asymmetric PCR 15-8Alternate Protocol 1: Generating Single:Stranded Template for Dideoxy Sequencing by

Single-Primer Reamplification •"'"•'' ; '' ••-.-, . 15-9Alternate Protocol 2: Preparing Double-Stranded PCR Products for Dideoxy Sequencing > 15-10Alternate Protocol 3: Dideoxy Sequencing of Single Strands Generated by.A; Exonuclease

Digestion of Double-Stranded PCR Products . 15-10Basic Protocol 2: Labeling and Chemical Sequencing of PCR Products 15-11Alternate Protocol 4: Genomic Sequencing of PCR Products * ; ... 15-12

15.3 QuantitationofRareDNAbyPCR ,, 15-13

15.4 Enzymatic Amplification of RNA by PCR 15-16Basic Protocol: PCR Amplification of RNA Under Optimal Conditions > ' 15-16Alternate Protocol 1: Avoiding Lengthy Coprecipitation and Annealing Steps ,, .-15-17Alternate Protocol 2: Introducing cDNA Directly into the Amplification Step . 15-18Support Protocol: Rapid Precipitation of Crude RNA 15-18

15.5 Ligation-Mediated PCR for Genomic Sequencing and Footprinting . , 15-19Basic Protocol: Ligatiori-Mediated Single-Sided PCR ' • '. ' ; 15-19Support Protocol 1: Preparation of Genomic DNA from Monolayer

Cells for DMS Footprinting 15-23Support Protocol 2: Preparation of Genomic DNA from Suspension

Cells for DMS Footprinting 15-26Support Protocol 3: Preparation of Genomic DNA for Chemical Sequencing , , 15-27

15.6 cDNA Amplification Using One-Sided (Anchored) PCR . 15-29Basic Protocol 1: Amplification of Regions Downstream (3') of Known Sequence 15-29Basic Protocol 2: Amplification of Regions Upstream (5') of Known Sequence ' 15-32

15.7 Molecular Cloning of PCR Products • - • IS-MBasic Protocol: Generation of T-A Overhangs • " • 15-34Alternate Protocol 1: Generation of Half-Sites 15-35

15.8 Differential Display of mRNA by PCR . 15-37


CONTENTS Page xvii

16 PROTEIN EXPRESSION

I N T R O D U C T I O N , 16-1

16.1 O v e r v i e w of P r o t e i n E x p r e s s i o n in E . coli ..''"'' 16 -2,.r • . General Strategy for Gene Expression in E. coli , i 16-3

Specific Expression Scenarios "' 16-3, Troubleshooting Gene Expression ' ' ' 16-4

16.2 Expression Using the T7 RNA Polymerase/Promoter System \ . 16-5Basic Protocol: Expression Using the Two-Plasmid System ,. 16-5Alternate Protocol 1: Selective Labeling of Plasmid-Encoded Proteins * 16-7Alternate Protocol 2: Expression by Infection with M13 Phage mGPl-2 " 16-8

16.3 Introduction to Expression by Fusion Protein Vectors .,, . 16-9S o l u b i l i t y o f t h e E x p r e s s e d P r o t e i n » ••••... . .- ; 1 6 - 9S t a b i l i t y o f t h e E x p r e s s e d P r o t e i n . . . . . . , . r •-?;•<-- " 1 6 - 1 0C l e a v a g e o f F u s i o n P r o t e i n s t o R e m o v e t h e C a r r i e r . . . . 1 6 - 1 0

1 6 . 4 E n z y m a t i c a n d C h e m i c a l C l e a v a g e o f F u s i o n P r o t e i n s 1 6 - 1 1Basic Protocol 1: Enzymatic Cleavage of Fusion Proteins with Factor Xa 16-11

' Support Protocol: Denaturing a Fusion Protein for Factor Xa Cleavage 16-12Alternate Protocol 1: Enzymatic Cleavage of Fusion Proteins with Thrombin 16-13Alternate Protocol 2: Enzymatic Cleavage of Matrix-Bound GST Fusion Proteins 16-13Alternate Protocol 3: Enzymatic Cleavage of Fusion Proteins with Enterokinase 16-14Basic Protocol 2: Chemical Cleavage of Fusion Proteins Using Cyanogen Bromide 16-15Alternate Protocol 4: Chemical Cleavage of Fusion Proteins Using Hydroxlamine 16-16Alternate* Protocol 5: Chemical Cleavage of Fusion Proteins by Hydrolysis at Low pH 16-16

16.5 Expression and Purification of Maltose-Binding Protein Fusions • 16-17Basic Protocol: Construction, Expression, and Purification of MBP Fusion-Proteins • .* ' . . • 16-18

, Support Protocol 1: Pilot Experiment to Characterize the Behavior of an MBP Fusion Protein 16-20Alternate.Protocol: Purification of Fusion Proteins from the Periplasm. • , •-• , 16-22Support Protocol 2: Purifying the Cleaved Protein by Ion Exchange Chromatography 16-22Support Protocol 3,:,. Pjirifying the Cleaved Protein by Affinity Chromatography-, ; , 16-23

16.6 Expression and Purification of Gliitathione-S-Transferase Fusion* ' - . '• yProteins ~ ' ; 16-24

16.7 Expression and Purification of Thioredoxin Fusion Proteins • , - - , . . , . , , . , 16-27Basic Protocol: Construction and Expression of a Thioredpxin Fusion Protein 16-27Support Protocol 1: E. coli Lysis Using a French Pressure Cell ' '"' '"• 16-29Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins , . 16-31Support Protocol 3: Purification of Thioredoxin Fusion Proteins by Heat Treatment 16-32

16.8 Overview of the Baculovirus Expression System •"' 16-33Baculovirus Expression System ' :; • 16-33Post-Translational Modification of Proteins in Insect Cells " ' 16-35Steps for Overproducing Proteins Using the Baculovirus Expression System < 16-35Choosing a Baculovirus Transfer Vector: Choosing a Baculovirus DNA . 16-35Choosing a Baculovirus DNA ' , , 16-38Reagents, Solutions, and Equipment for the Baculovirus Expression System 16-38

16.9 Maintenance of Insect Cell Cultures and Generation of Recombinant ;

Baculoviruses •..•••••» 16-39

Basic Protocol 1: Maintenance and Culture of Insect Cells ' ' " "* 16-40Basic Protocol 2: Cotransfection of Insect Cells Using Linearized Baculoviral DNA 16-41Alternate Protocol 1: Generation of Recombinant Baculovirus Using Wild-Type Baculoviral DNA 16-43Basic Protocol 3: Preparation of Baculovirus Stocks 16-45Basic Protocol 4: Titering Baculovirus Stocks Using Plaque Assay 16-47

16.10 Expression and Purification of Recombinant Proteins Using thef Baculovirus System . 16-48

Basic Protocol 1: Small-Scale Expression for Initial Analysis 16-49Support Protocol 1: Determining Time Course of Maximum Protein Production 16-50Support Protocol 2: Metabolic Labeling of Recombinant Proteins 16-51

Short Protocols in Molecular Biology .

Pagexviii CONTENTS

Basic Protocol 2: Large-Scale Production of Recombinant Proteins •,'•'• •••< "• " - . , 16-52

Basic Protocol 3: Purification of Recombinant Proteins Containing a Polyhistidine (6xHIS) Tag 16-53Alternate Protocol: Purification of Recombinant Proteins Containing a GST Tag * 16-54

16.11 Overview of Protein Expression in Mammalian Cells • ' • - '16-56

Viral-Mediated Gene Transfer " ,-,-..« : , : " « ....-.' 16-56Transient Expression , , . 16-56Stable DNA Transfection . . , , . . 16-57

r Ariiplif icationofTransfectedDNA ,, , . , • • • • • • 16-59Expression Vectors 16-59Choice of Expression System * 16-60Troubleshooting : 16-60

16.12 Transient Expression of Proteins Using COS Cells I,, , 16-60

16.13 Overview of the Vaccinia Virus Expression System •!•-. '•'"•'; 16-63

Vaccinia Replication Cycle ,•• . • 16-63Effects of Vaccinia Infection , . . • 16-64Vaccinia Vector Expression System ..,,.-• , . 16-65

I Steps For Expression of Genes Using Vaccinia Vectors , . . ; .,,<• 16-66Safety Precautions for Using Vaccinia . • •, v < , r , • 16-66

16.14 Preparation of Cell Cultures and Vaccinia Virus Stocks • i ' 16-67

Basic Protocol 1: Culture of Monolayer Cells- , ,. . 16-68

Basic Protocol, 2: Culture of Cells in Suspension . . . . . , , iC • • " . . , . 16-69

Basic Protocol 3: Preparation of a Vaccinia.Virus Stock , , to;c <' 16-70Support.Protpcol l :Titrat ion of Vaccinia Virus Stocks by Plaque Assay , : . • . 16-70Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts . •< , , 16-71Basic Protocol 5: Preparation of an MVA Stock ' . . ., . ;. 16-72Support Protocol 2: Titration of MVA Stocks by Immunostaining 16-73

16.15 Generation of Recombinant Vaccinia Viruses : . 16-75

Basic Protocol 1: Transfection.of Infected Cells with a Vaccinia Vector ,' '. • , . . . . ,., 16-75

Support Protocol 1: Purification of Vaccinia, Virus , . '• • •• 16-79

Support Protocol 2: Isolation of Vaccinia Virus DNA . . . , 16-81Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques. ., 16-82Basic Protocol 3: Amplification o f ^ P l a q u e . . . " -. 16-84Basic Protocol 4: Live Immunostaining of MVA Recombinants . J,,,. % . . 16-85Support Protocol 3: Coating Plates with Concanayalin A -;. >'. '•', ,.' . 16-87

1 6 . 1 6 C h a r a c t e r i z a t i o n . o f R e c o m b i n a n t V a c c i n i a V i r u s e s a n d T h e i r P r o d u c t s • ' . < > . : • ; 1 6 - 8 7

Basic Protocobl: Detection of Vaccinia DNA.Using PGR s-; ' ." • - ;;u 16-88Basic Protocol 2: Detection of Vaccinia DNA.Using Southern Blot Hybridization . 16-90Basic Protocol 3: Detection of Vaccinia DNA UsingX>ot-Blot Hybridization , r16-91Alternate Protocol: Detection of Expressed.Protein by a dot-Blot Procedure . 16-92Basic Protocol 4: Detection of Expressed Protein Using Immunoblottirig . . ! , , ; . 16-93Basic Protocol 5: Detection of.Expr'essed Protein Using Immunoprecipitation ~ ,,. 16-94

16.17 Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase :>• ,r ;

Hybrid System 16-95

Basic Protocol 1: Liposorhe-Mediated Transfe'ction Following Recombinant Vaccinia Virus

(VTF7-3) Infection > , , , • . • • ,16-95Basic Protocol 2: Coinfection with Two Recombinant Vaccinia Viruses : 16-98Basic Protocol 3: Infection of OST7-1 cells;.with a ;Singl&Virus • . . 16-100Basic Protocol 4: Gene Expression Using the Vote System ,. • . ; . - . , *. • 16-100Support Protocol: Detection of Expressed Protein Using Pulse Labeling : ; •, 16-101

1 16.18 Inducible Gene Expression Using an Autoregulatory, Tetracycline- < '

Controlled System 16-102

Basic Protocol: Calcium Phosphate-Mediated Stable Transiectibn of NIH3T3 Cells with 'pTet-tTAk and Tetracyline-Regulated Target Plasmids' ' - : ;• 16-103

Support Protocol: Analysis of Target Gene Protein Expression 16-107

CONTENTS


Page xix

17 ANALYSIS OF PROTEIN PHOSPHORYLATION

INTRODUCTION . 17-1

,17.1 Overview of Protein Phosphorylation • \ • 17-1

•.-'"• 17.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for' • Immunoprecipitation > 17-3

Basic Protocol: Labeling Cultured Cells with T>i and Lysis Using Mild Detergent 17-3' r Alternate Protocol: Lysis of Cells by Boiling in SDS ' •' 17-5

17.3 Phosphoamino Acid Analysis , 17-6Basic Protocol: Acid Hydrolysis and Two-Dimensional Electrophoretic

Analysis of Phosphoamino Acids . 17-6Alternate Protocol: Alkali Treatment to Enhance Detection of Tyr- and Thr-Phosphorylated

Proteins Blotted onto Filters : 17-9

17.4 Analysis of Phosphorylation of Unlabeled Proteins 17-10Basic Protocol 1: Immunoblotting with Anti-Phosphotyrosine

Antibodies and Detectiion Using [ T]ProteinA. 17-10Alternate Protocol: Detection of Bound Antibodies by Enhanced Chemiluminescence (ECL) 17-11Basic Protocol 2: Identification of Phosphorylated Proteins by

Phosphatase Digestion . . . - . • • 17-12

17.5 Detection of Phosphorylation by Enzymatic Techniques • 17-13Basic Protocol 1: Digestion of Phosphoproteins with Nonspecific Acid Phosphatases ' ' 17-14Alternate Protocol 1: Digestion of Phosphoproteins with Nonspecific Alkaline Phosphatase 17-15Basic Protocol 2: Digestion of Phosphoproteins with Protein Seririe/Threonine'Phosphatases 17-15Alternate Protocol 2: Digestion of Phosphoproteins with Protein Tyrosirie Phosphatases" 17-16Support Protocol: Measurement and Identification of Released T> 17-16

17.6 Assays of Protein Kinases Using Exogenous Substrates 17-17Strategic Planning • 17-17B asic Protocol 1: Assay for Cyclic Nucleotide-Dependent Protein Kinases 17-19Basic Protocol 2: Assay for Protein Kinase C Isoforms ' ' 17-20Basic Protocol 3: Assay for Casein Kinases Using P-Casein ' ; 17-20Alternate Protocol: Assay for Casein Kinases Using a Peptide Substrate 17-21Basic Protocol 4: Assay for Ca +/Calmodulin-Dependent Kinases " ' ' 17-22Basic Protocol 5: Assay for Tyrosine Kinases • , - 17-23Basic Protocol 6: In-Gel Protein Kinase Assays ' ' 17-24Support Protocol 1: Preparing a Cell Lysate for Kinase Assays . '« : 17-25Support Protocol 2: TCA Precipitation to Determine Incorporation of Radioactivity •: i 17-25Support Protocol 3: Adsorption onto p81 Phosphocellulose Paper '• • -;?n' 17-26

17.7 Permeabilization Strategies to Study Protein Phosphorylation 17-28B asic Protocol 1: Analysis of Protein Phosphorylation in Permeabilized Cells '" .' . 17-28Intact Cell Sample Preparation for Electrophoretic Analysis of Protein Phosphorylation ' ; 17-31

Alternate Protocol 1: Intact Cell Sample Preparation for SDS-PAGE ' • 17-31Alternate Protocol 2: Intact Cell Sample Preparation for Isoelectric Focusing ~> ." i.s \l-7>2

18 SEQUENCE SIMILARITY SEARCHING USING THE BLAST - :FAMILY OF PROGRAMS

18.1 Sequence Similarity Searching Using the BLAST Family of Programs • -•'- 18-1A c c e s s i n g B L A S T P r o g r a m s a n d D o c u m e n t a t i o n : . > : • ; . . ••. - .. • 18-1Introduction to BLAST '• ' 18-2Examples of BLAST Searches , ,r . ;„ . -18-6Searching Strategies 18-19Appendix A: BLAST Parameters . • j ,• . . . 18-22Appendix B: Sequence Identifier Syntax . . 18-23


Page xx CONTENTS

19 ANALYSIS OF PROTEIN INTERACTIONS

INTRODUCTION 19-1

19.1 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins 19-5Basic Protocol 1: Characterizing a Bait Protein 19-5Basic Protocol 2: Performing an Interactor Hunt 19-13Alternate Protocol 1: Rapid Screen for Interaction Trap Positives 19-20Alternate Protocol 2: Performing a Hunt by Interaction Mating . 19-22Support Protocol 1: Preparation of Protein Extracts for Immunoblot Analysis 19-24Support Protocol 2: Preparation of Sheared Salmon Sperm Carrier DNA 19-25

19.2 Affinity Purification of Proteins Binding to GST Fusion Proteins 19-26Basic Protocol: GST Fusion Protein-Affinity Purification ' 19-26Support Protocol: Preparation of E. coli Extract 19-28

19.3 Phage-Based Expression Cloning to Identify Interacting Proteins 19-29Strategic Planning 19-29Basic Protocol: Interaction cloning 19-31

APPENDICES

Al Reagents and Solutions - Al-1

A2 Standard Measurements and Data " • A2-1Useful Measurements and Data A2-1Characteristics of Nucleic Acids A2-6Radioactivity - A2-12Centrifuges and Rotors , . A2-15

A3 Commonly Used Techniques in Biochemistry and Molecular Biology A3-13 A Autoradiography • ' • A3-13B Silanizing, Glassware . A3-33C Dialysis and Ultrafiltration ..'.. A3-43D Quantitation of DNA and RNA with Absorption and . ;

Fluorescence Spectroscopy .. , A3-103E Introduction of Restriction Enzyme Recognition Sequences

by Silent Mutation A3-123F Techniques for Mammalian Cell Tissue Culture .. „ A3-143G Safe Use of Radioisotopes A3-223H Statistics for the Molecular Biologist: Group Comparisons . A3-33

A4 Selected Suppliers of Reagents and Equipment A4-1

A5 References • • . • • . . A5-1

INDEX


CONTENTS Page xxi

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