Short protocols in molecular biology: a compendium of methods from "Current protocols in molecular biology"
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| Format: | Book |
|---|---|
| Language: | English |
| Published: |
New York [u.a.]
Wiley
1999
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| Edition: | 4. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | Getr. Zählung Ill., graph. Darst. |
| ISBN: | 047132938X |
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| 245 | 1 | 0 | |a Short protocols in molecular biology |b a compendium of methods from "Current protocols in molecular biology" |c ed. by Frederick M. Ausubel ... |
| 250 | |a 4. ed. | ||
| 264 | 1 | |a New York [u.a.] |b Wiley |c 1999 | |
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| adam_text | CONTENTS
xxiii Preface
xxvii Contributors
1 ESCHERICHIA COLI, PLASMIDS, AND BACTERIOPHAGES
INTRODUCTION 1 1
1.1 Media Preparation and Bacteriological Tools 1 2
Minimal Media 1 2
Rieh Media 1 3
Solid Media 1 3
TopAgar 1 4
StabAgar 1 4
Tools 1 4
1.2 Growth in Liquid Media 1 5
Basic Protocol 1: Growing an Overnight Culture 1 5
Basic Protocol 2: Growing Larger Cultures 1 5
Basic Protocol 3: Monitoring Growth 1 6
1.3 Growth on Solid Media 1 6
Basic Protocol 1: Titering and Isolating Bacterial Colonies by Serial Dilutions 1 6
Basic Protocol 2: Isolating Single Colonies by Streaking a Plate 1 7
Basic Protocol 3: Isolating Single Colonies by Spreading a Plate 1 7
Support Protocol 1: Replica Plating 1 8
Support Protocol 2: Strain Storage and Revival 1 8
1.4 Selected Topics from Classical Bacterial Genetics 1 9
1.5 Maps of Plasmids 1 13
Choosing a Plasmid Vector 1 15
1.6 Miniprepsof Plasmid DNA 1 22
Basic Protocol 1: Alkaline Lysis Miniprep 1 22
Alternate Protocol: Alkaline Lysis in 96 Well Microtiter Dishes 1 22
Basic Protocol 2: Boiling Miniprep 1 23
Support Protocol: Storage of Plasmid DNA 1 24
1.7 Large Scale Preparation of Plasmid DNA 1 24
Preparation of Crude Lysates 1 24
Basic Protocol 1: Alkaline Lysis 1 24
Basic Protocol 2: CsCl/Ethidium Bromide Equilibrium Centrifugation 1 25
Alternate Protocol: Plasmid DNA Purification by Anion Exchange or Size Exclusion
Chromatography 1 26
1.8 Introduction of Plasmid DNA into Cells 1 27
Basic Protocol 1: Transformation Using Calcium Chloride 1 27
Alternate Protocol: One Step Preparation and Transformation of Competent Cells 1 28
Basic Protocol 2: High Efficiency Transformation by Electroporation 1 29
1.9 Introduction to Lambda Phages 1 30
Lytic Growth 1 31
Lysogenic Growth 1 32
1.10 Lambda as a Cloning Vector 1 33
Advantages of Using Lambda 1.33
Selections for Inserted DNA 1.33
Maps of Lambda Derived Cloning Vectors 1 36
1.11 Plating Lambda Phage to Generate PIaques 1 36
Basic Protocol 1: Isolating a Single Plaque by Titering Serial Dilutions 1 36
Basic Protocol 2: Phage Transfection and In Vitro Packaging 1 37
. Short Protocols in Molecular Biology
S*/MklT EkJTC _
1.12 Growing Lambda Derived Vectors l_3g
Basic Protocol: Making a Stock of Phage by Plate Lysis 1.38
Alternate Protocol: Making a Liquid Lysate l_3g
1.13 Preparing Lambda DNA from Phage Lysates I.39
Basic 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 I.44
Basic Protocol: Preparing Single Stranded DNA from Plasmids Using Helper Phage 1 44
PREPARATION AND ANALYSIS OF DNA
INTRODUCTION 2 1
2.1 A Purification and Concentration of DNA from Aqueous Solutions 2 3
Basic Protocol: Phenol Extraction and Ethanol Precipitation of DNA 2 3
Alternate Protocol 1: Precipitation of DNA Using Isopropanol 2 4
Support Protocol 1: Buffering Phenol and Preparing Phenol/ Chloroform/Isoamyl Alchohol 2 4
Support Protocol 2: Concentration of DNA Using Butanol 2 5
Support Protocol 3: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction 2 5
Alternate Protocol 2: Purification of DNA Using Glass Beads 2 6
Alternate Protocol 3: Purification and Concentration of RNA and Dilute Solutions of DNA 2 6
Alternate 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 10
Basic Protocol: Preparation of Plant DNA Using CsCl Centrifugation 2 10
Alternate Protocol: Preparation of Plant DNA Using CTAB 2 11
2.4 Preparation of Genomic DNA from Bacteria 2.i2
Basic Protocol: Miniprep of Bacterial Genomic DNA 2 12
Alternate Protocol: Large Scale CsCl Prep of Bacterial Genomic DNA 2 13
Support Protocol: Removal of Polysaccharides from Existing Genomic DNA Preps 2 14
2.5A Agarose Gel Electrophoresis
Basic Protocol: Resolution of Large DNA Fragments on Agarose Gels 2 14
Support Protocol: Minigels and Midigels
2.5B Pulsed Field Gel Electrophoresis
Basic Protocol: Field Inversion Electrophoresis
Alternate Protocol: Chef Electrophoresis
Support Protocol: Preparation of High Molecular Weight DNA Samples and Size Markers 2 Jg
2.6 Isolation and Purification of Large DNA Restriction Fragments
from Agarose Gels
Basic Protocol 1: Electroelution from Agarose Gels °
Basic Protocol 2: Electrophoresis onto NA 45 Paper 2~20
B asic Protocol 3: Isolation of DNA Fragments Using Low 2 21
Gelling/Melting Temperature Agarose Gels
Alternate Protocol 1: Recovery of DNA from Low Gelling/Melting 2 22
Temperature Agarose Gels Using p Agarase Digestion
Alternate Protocol 2: Recovery of DNA from Low Gelling/Melting
Temperature Agarose Using Glass Beads
Support Protocol: Rapid Estimation of DNA Concentration by 2 23
Ethidium Bromide Dot Quantitation
2.7 Separation of Small DNA Fragments by Conventional Gel Electrophones Vt
Bas1C Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis
Short Protocols in Molecular Biology
2.8 Capillary Electrophoresis of DNA 2 28
Instrumentation 2 28
Separation Theory 2 29
Strategic Planning 2 29
Basic Protocol 1: Separation of Oligonucleotides 2 31
Basic Protocol 2: Quantitative PCR Analysis 2 33
Alternate 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 34
Support Protocol: Calibration of a UV Transilluminator 2 37
Alternate Protocol 1: Southern Blotting onto a Nylon Membrane
with an Alkaline Buffer 2 37
Alternate Protocol 2: Southern Blotting by Downward Capillary Transfer 2 38
Alternate Protocol 3: Electroblotting from a Polyacrylamide
Gel to a Nylon Membrane 2 38
2.9B Dot and Slot Blotting of DNA 2 39
Basic Protocol: Dot and Slot Blotting of DNA onto Uncharged
Nylon and Nitrocellulose Membranes Using a Manifold 2 40
Alternate Protocol 1: Dot and Slot Blotting of DNA onto a
Positively Charged Nylon Membrane Using a Manifold 2 41
Alternate Protocol 2: Manual Preparation of a DNA Dot Blot 2 41
2.10 Hybridization Analysis of DNA Blots 2 42
Basic Protocol: Hybridization Analysis of a DNA Blot with a
Radiolabeled DNA Probe 2 42
Alternate Protocol: Hybridization Analysis of a DNA Blot with a
Radiolabeled RNA Probe 2 44
Support 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 2
Basic Protocol: Digesting a Single DNA Sample with a Single
Restriction Endonuclease 3 2
Alternate Protocol 1: Digesting DNA with Multiple Restriction Endonucleases 3 3
Alternate Protocol 2: Digesting Multiple Samples of DNA 3 6
Alternate Protocol 3: Partial Digestion of DNA with Restriction Endonucleases 3 7
Support Protocol: Methylation of DNA 3 7
3.2 Mapping by Multiple Endonuclease Digestions 3 8
3.3 Mapping by Partial Endonuclease Digestions 3 9
3.4 Reagents and Radioisotopes Used to Manipulate Nucleic Acids 3 9
Stock Solutions 3 9
lOx Enzyme Buffers 3 10
Enzyme Reaction Conditions and Applications 3 13
Nucleoside Triphosphates 3 13
Radioisotopes for Labeling Nucleic Acids 3 16
Basic Protocol: Measuring Radioactivity in DNA and RNA by Acid Precipitation 3 16
Alternate Protocol: Spin Column Procedure for Separating
Radioactively Labeled DNA from Unincorporated dNTP Precursors 3 17
3.5 DNA Dependent DNA Polymerases 3 18
Klenow Fragment of E. coli DNA Polymerase I 3 18
Basic Protocol 1: Labeling the 3 Ends of DNA 3 19
Short Protocols in Molecular Biology
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Basic Protocol 2: Repairing 3 or 5 Overhanging Ends to Generat
Blunt Ends 3 20
Basic Protocol 3: Labeling of DNA by Random Oligonucleotide
Primed Synthesis 3 20
T4 DNA Polymerase 3 21
Native T7 DNA Polymerase 3 23
Modified T7 DNA Polymerase 3 23
Taq DNA Polymerase 3 24
3.6 Template Independent DNA Polymerases 3 25
3.7 RNA Dependent DNA Polymerases 3 26
3.8 DNA Dependent RNA Polymerases 3 27
3.9 DNA Independent RNA Polymerases 3 28
3.10 Phosphatases and Kinases 3 28
Alkaline Phosphatases: BAP and CIP 3 28
T4 Polynucleotide Kinase 3 29
Basic Protocol 1: Labeling 5 Ends by the Forward Reaction 3 29
Basic Protocol 2: Labeling 5 Termini by the Exchange Reaction 3 29
3.11 Exonucleases 3 30
Single Stranded 5 3 and 3 — 5 Exonucleases 3,39
Double Stranded 5 3 Exonucleases 3.3O
Double Stranded 3 5 Exonucleases 3.39
3.12 Endonudeases 3.3I
Bal3 Nuclease 3.31
SI Nuclease 3.32
Mung Bean Nuclease 3.32
Micrococcal Nuclease 3.33
Deoxyribonuclease I (DNase I) 3.33
3.13 Ribonucleases 3.34
Ribonuclease A 3.34
Ribonuclease H 3.34
Ribonuclease Tl 3 3,5
3.14 DNA Ligases 3.36
T4 DNA Ligase 3 36
E. coli DNA Ligase 3 36
3.15 RNA Ligases 3 36
T4 RNA Ligase 3 36
3.16 Subcloning of DNA Fragments
Basic Protocol
Alternate Protocol: Ligation of DNA Fragments in Gel Slices 3 3o
3.17 Constructing Recombinant DNA Molecules by the Polymerase
Chain Reaction
Basic Protocol: Subcloning DNA Fragments
3.18 Labeling and Colorimetric Detection of Nonisotopic Probes
Basic Protocol 1: Preparation of Biotinylated Probes by
Nick Translation
Basic Protocol 2: Preparation of Biotinylated Probes by Random Oligonucleotide Primed Synthesis 3 45
Support Protocol: Colonmetnc Detection of Biotinylated Probes ^ mesis j 4D
Alternate Protocol: Preparation and Detection of Digoxigenin
Labeled DNA Probes
3 47
Short Protocols in Molecular Biology Page vi —
3.19 Chemiluminescent Detection of Nonisotopic Probes 3 47
Basic Protocol: Chemiluminescent Detection of Biotinylated Probes 3 47
Alternate Protocol: Chemiluminescent Detection of Digoxigenin Labeled Probes 3 50
Support 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 2
Basic Protocol 4 2
Support Protocol: Removal of Contaminating DNA 4 3
4.2 Guanidinium Methods for Total RNA Preparation 4 4
Basic Protocol: Single Step RNA Isolation from Cultured Cells or Tissues 4 4
Alternate Protocol 1: CsCl Purification of RNA from Cultured Cells 4 5
Alternate Protocol 2: CsCl Purification of RNA from Tissue 4 6
4.3 PhenoI/SDS Method for Plant RNA Preparation 4 7
4.4 Preparation of Bacterial RNA 4 8
Basic Protocol 1: Isolation of High Quality RNA from Gram Negative Bacteria 4 8
Basic Protocol 2: Isolation of RNA from Gram Positive Bacteria 4 10
Alternate 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 12
Basic Protocol: SI Analysis of mRNA Using M13 Template 4 12
Alternate Protocol 1: Synthesis of Single Stranded Probe from
Double Stranded Plasmid Template 4 15
Alternate Protocol 2: Quantitative SI Analysis of mRNA Using
Oligonucleotide Probes 4 15
Support Protocol: Controls for Quantitative S1 Analysis of mRNA 4 16
4.7 Ribonuclease Protection Assay 4 17
Basic Protocol 4 17
Support Protocol 1: Gel Purification of RNA Probes 4 18
Support Protocol 2: Preparation of Template DNA 4 19
4.8 Primer Extension 4 19
4.9 Analysis of RNA by Northern and Slot Blot Hybridization 4 21
Basic Protocol: Northern Hybridization of RNA Fractionated by Agarose Formaldehyde Gel
Electrophoresis 4 22
Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment 4 24
Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting 4 25
Support 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 2
Representation and Randomness 5 3
Subgenomic Libraries 5 3
Vectors 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
Short Protocols in Molecular Biology
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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 6
Basic Protocol: Hybridization in Formamide 6 6
Alternate Protocol: Hybridization in Aqueous Solution 6 6
6.4 Using Synthetic Oligonucleotides as Probes 6 7
Basic Protocol 1: Hybridization in Sodium Chloride/Sodium Citrate (SSC) 6 7
Basic Protocol 2: Hybridization in Tetramethylammonium Chloride (TMAC) 6 8
Support Protocol: Labeling the 5 Ends of Mixed Oligonucleotides 6 10
6.5 Purification of Bacteriophage Clones 6 11
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 Xgtl 1 Expression Library with Antibodies 6 12
Alternate Protocol: Induction of Fusion Protein 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
Generating YAC Libraries 6 16
YAC Library Screening by a Core Laboratory 6 16
Designing a Locus Specific PCR Assay for Screening 6 18
Analyzing Individual YAC Clones 6 18
Construction and Analysis of a YAC Insert Sublibrary 6 19
6.10 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 20
Basic Protocol 3: Preparation of Yeast Chromosomes in Agarose
Plugs for Pulsed Field Gel Electrophoresis 6_22
Basic Protocol 4: End Fragment Analysis Using PCR Amplification 6 23
Alternate Protocol: End Fragment Analysis by Subcloning into a
Bacterial Plasmid Vector g_26
Support Protocol: Design and Preparation of pUC19 ES and pUC19 HS Subcloning Vector 6 28
Basic Protocol 5: Preparation of High Molecular Weight YAC Containing Yeast DNA in Solution 6 28
Basic Protocol 6: Preparation and Analysis of a YAC Insert Sublibrary 6 30
r DNA SEQUENCING
OVERVIEW OF DNA SEQUENCING METHODS 7_j
7.1 DNA Sequencing Strategies 7g
Dideoxy Sequencing _ „
Chemical Sequencing _ ~
7.2 Constructing Nested Deletions for Use in DNA Sequencing 7_U
Basic Protocol 1: Using Exonuclease ID to Construct Unidirectional Deletions 7.11
Support Protocol 1: Protection of DNA from Exonuclease m
Digestion Using [ot35S]dNTPs _ j
Basic Protocol 2: Using Bal 31 Nuclease to Construct Nested Deletions 7!l5
Support Protocol 2: Preparation of M13mp Sequencing Vector DNA for Subclonine of
Bal 31 Digested DNA Fragments ? 20
Short Protocols in Molecular Biology Page viii ~ ^
7.3 Preparation of Templates for DNA Sequencing 7 21
Basic Protocol 1: Preparation of Single Stranded M13 Phage DNA 7 21
Basic Protocol 2: Preparation of A. DNA from Small Scale Lysates 7 22
Basic Protocol 3: Miniprep of Double Stranded Plasmid DNA
for Dideoxy Sequencing 7 23
Basic Protocol 4: Alkali Denaturation of Double Stranded Plasmid
DNA for Dideoxy Sequencing 7 24
Basic 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 25
Basic Protocol 1: Labeling/Termination Sequencing Reactions Using Sequenase (Modified T7
DNA Polymerase) 7 26
Alternate Protocol 1: Using Mn + in the Labeling/Termination Reactions 7 30
Alternate Protocol 2: Using Taq DNA Polymerase in the Sanger Procedure 7 30
Alternate Protocol 3: One Step Sequencing Reactions Using 5 End Labeled Primers 7 31
Basic Protocol 2: Thermal Cycle Sequencing Reactions Using
oc Labeled Nucleotides 7 32
Alternate Protocol 4: Thermal Cycle Sequencing Reactions Using
5 End Labeled Primers 7 33
Alternate Protocol 5: Cycle Sequencing Using Fluorescence Dye Labeled Primer (Dyeprimer)
or Terminator (Dyeterminator) 7 35
7.5 Dideoxy DNA Sequencing with Chemiluminescent Detection 7 37
Basic Protocol: DNA Sequencing Using Biotinylated Primers with Chemiluminescent Detection 7 38
Alternate Protocol 1: Two Step (Indirect) Detection Using
Streptavidin and Biotinylated Alkaline Phosphatase 7 40
Alternate Protocol 2: Sequencing with Hapten Labeled Primers
and Detection with Antibody Alkaline Phosphatase Conjugates 7 41
7.6 Denaturing Gel Electrophoresis for Sequencing 7 42
Basic Protocol: Pouring, Running, and Processing Sequencing Gels 7 43
Alternate Protocol 1: Buffer Gradient Sequencing Gels 7 46
Alternate Protocol 2: Electrolyte Gradient Sequencing Gels 7 46
Alternate Protocol 3: Formamide Containing Sequencing Gels 7 47
7.7 Computer Manipulation of DNA and Protein Sequences 7 47
Sequence Data Entry 7 48
Sequence Data Verification and Assembly 7 50
Restriction Mapping 7 52
Prediction of Nucleic Acid Structure 7 54
Oligonucleotide Design Strategy 7 55
Identification of Protein Coding Regions 7 57
Homology Searching 7 58
Genetic Sequence Databases and Other Electronic Resources
Available to Molecular Biologists 7 60
Appendix 7 60
8 MUTAGENESIS OF CLONED DNA
INTRODUCTION 8 1
8.1 Oligonucleotide Directed Mutagenesis Without Phenotypic Selection 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 8 8
8.3 Region Specific Mutagenesis 8 9
Basic Protocol 8 9
Support Protocol: Enrichment of Mutant Clones 8 12
___^ Short Protocols in Molecular Biology
8.4 Linker Scanning Mutagenesis of DNA 8 12
Basic 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 15
Basic Protocol 1: Introduction of Restriction Endonuclease
Sites by PCR 8 15
Basic Protocol 2: Introduction of Point Mutations by PCR 8 16
Alternate Protocol: Introduction of a Point Mutation by
Sequential PCR Steps 8 21
9 INTRODUCTION OF DNA INTO MAMMALIAN CELLS
OVERVIEW OF TRANSFECTION METHODS 9 1
9.1 Calcium Phosphate Transfectton 9 6
Basic Protocol: Transfection Using Calcium Phosphate DNA
Precipitate Formed in HEPES 9 6
Support Protocol 1: Glycerol/DMSO Shock of Mammalian Cells 9 7
Alternate Protocol: High Efficiency Transfection Using Calcium Phosphate DNA Precipitate
Formed in BES 9 8
9.2 Transfection Using DEAE Dextran 9 9
Basic Protocol: General Procedure for DEAE Dextran Transfection 9 9
Alternate Protocol 1: Experiment: Transfection to Test Enzyme Structure/Activity Relationships 9 11
Support Protocol: Charcoal Stripping of Fetal Bovine Serum 9 12
9.3 Transfection by Electroporation 9 13
Basic Protocol: Electroporation into Mammalian Cells 9 13
Alternate Protocol: Electroporation into Plant Protoplasts 9 14
9.4 Liposome Mediated Transfection 9 15
Basic Protocol: Transient Expression Using Liposomes 9 15
Alternate Protocol: Stable Transformation Using Liposomes 9 16
9.5 Selection of Transfected Mammalian Cells: Strategic Planning 9 16
Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells 9 19
Basic Protocol 2: Selectable Markers for Mammalian Cells 9 20
9.6 Overview of Genetic Reporter Systems 9 24
Design of Reporter Vectors 9 26
In Vitro Reporter Assays 9 26
In Vivo Reporter Assays 9 29
9.7A Isotopic Assay for Reporter Gene Activity 9 33
Basic Protocol 1: Chromatographic Assay for CAT Activity 9 33
Alternate Protocol 1: In Situ Lysis of Cells for CAT Assay 9 35
Alternate Protocol 2: Phase Extraction Assay for CAT Activity 9 36
Basic Protocol 2: Radioimmunoassay for Human Growth Hormone 9 37
9.7B Nonisotopic Assays for Reporter Gene Activity 9 38
Basic Protocol 1: Firefly Luciferase Reporter Gene Assay 9 38
Alternate Protocol: Luciferase Assay in Freeze Thaw Lysed Cells 9 39
Basic Protocol 2: Chemiluminescent P Galactosidase Reporter Gene Assay 9 40
9.8 Overview of the Retrovirus Transduction System 9 41
Retrovirus Life Cycle 9 41
Replication Incompetent Vectors 9.44
Replication Competent Vectors 9 46
Packaging Lines and Virus Production 9.45
Murine Retroviruses 9.5O
Avian Retroviruses 9,5 j
Safety Issues 9.52
9.9 Preparation of a Specific Retrovirus Producer Cell Line 9 52
Basic Protocol 1: Introduction of a Retrovirus Vector into a Packaging Cell Line 9 52
Basic Protocol 2: Determination of Viral Titer: Identification of
Producer Clones Making High Titer Virus 9 55
Alternate Protocol: Rapid Evaluation of Producer Colonies 9 56
Support 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 57
Support Protocol: Growth and Storage of 293 Cells 9 58
Basic Protocol 2: Infection of Adherent Cells with Retroviral Supernatant 9 59
Alternate Protocol 1: Infection of Adherent Cells by Spin Infection 9 60
Basic Protocol 3: Infection of Nonadherent Cells by Retroviral Supernatant 9 61
Alternate Protocol 2: Infection of Nonadherent Cells by Cocultivation 9 62
Alternate Protocol 3: Infection of Nonadherent Cells by Spin Infection 9 63
9.11 Large Scale Preparation and Concentration of Retrovirus Stocks 9 63
Basic Protocol: Preparation of Virus Stock and Concentration by Centrifugation 9 64
Alternate Protocol 1: Concentration by PEG Precipitation and Chromatography 9 65
Alternate Protocol 2: Concentration Using Molecular Weight Cutoff Filters 9 65
9.12 Detection of Helper Virus in Retrovirus Stocks 9 66
Basic Protocol: Detection of Helper Virus Through Horizontal Spread of Drug Resistance 9 66
Alternate Protocol 1: Proviral Rescue to Detect Replication Competent Retrovirus 9 67
Alternate Protocol 2: Reverse Transcriptase Assay to Detect Helper Virus 9 68
9.13 Retrovirus Infection of Cells In Vitro and In Vivo 9 69
Infection of Cells In Vitro 9 69
Infection of Rodents In Vivo 9 70
10 ANALYSIS OF PROTEINS
OVERVIEW OF PROTEIN PURIFICATION AND CHARACTERIZATION 10 1
10.1 Determination of Protein Concentration 10 8
Basic Protocol 1: Using A280to 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 10
Electricity and Electrophoresis 10 10
Basic Protocol 1: Denaturing (SDS) Discontinuous Gel
Electrophoresis: Laemmli Gel Method 10 11
Alternate Protocol 1: Electrophoresis in Tris Tricine Buffer Systems 10 16
Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers 10 18
Alternate Protocol 3: Continuous SDS PAGE 10 19
Alternate Protocol 4: Casting and Running Ultrathin Gels 10 19
Support Protocol 1: Casting Multiple Single Concentration Gels 10 20
Alternate Protocol 5: Separations of Proteins on Gradient Gels 10 22
Support Protocol 2: Casting Multiple Gradient Gels 10 25
Basic Protocol 2: Electrophoresis in Single Concentration Minigels 10 27
Support Protocol 3: Preparing Multiple Gradient Minigels 10 29
10.3 Two Dimensional Gel Electrophoresis Using the O Farrell System 10 30
Basic Protocol 1: First Dimension (Isoelectric Focusing) Gels 10 30
Basic Protocol 2: Second Dimension Gels 10 32
Isoelectric Focusing of Very Basic and Very Acidic Proteins 10 34
Alternate Protocol: Two Dimensional Minigels 10 35
Support Protocol: Solubilization and Preparation of Proteins in Tissue Samples 10 35
10.4 Staining Proteins in Gels 10 36
Basic Protocol 1: Coomassie Blue Staining 10 36
Basic Protocol 2: Silver Staining 10 36
Alternate Protocol: Nonammoniacal Silver Staining 10 3 /
Short Protocols in Molecular Biolo
* rU4TTWTS Page
Basic Protocol 3: Rapid Silver Staining 10 38
Support Protocol: Gel Photography 10 39
10.5 Immunoblotting and Immunodetection 10 40
Basic Protocol 1: Protein Blotting with Tank Transfer Systems 10 40
Alternate Protocol 1: Protein Blotting with Semidry Systems 10 42
Alternate Protocol 2: Blotting of Stained Gels 10 43
Support Protocol 1: Reversible Staining of Transferred Proteins 10 44
Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody 10 44
Alternate Protocol 3: Immunoprobing with Avidin Biotin Coupling
to the Secondary Antibody 10 45
Basic Protocol 3: Visualization with Chromogenic Substrates 10 46
Alternate Protocol 4: Visualization with Luminescent Substrates 10 46
Support Protocol 2: Stripping and Reusing Membranes 10 48
10.6 Gel Filtration Chromatography 10 48
Basic Protocol 1: Desalting (Group Separation) 10 48
Basic Protocol 2: Protein Fractionation 10 54
Basic Protocol 3: Determination of Molecular Size 10 58
Support Protocol: Column Calibration 10 60
10.7 Ion Exchange Chromatography 10 62
Strategic Planning 10 62
Basic Protocol 1: Batch Adsorption and Step Gradient Elution
with Increasing Salt Concentration 10 63
Basic Protocol 2: Column Chromatography with Linear Gradient Elution 10 66
Support Protocol 1: Test Tube Pilot Experiment to Determine Starting
Conditions for Ion Exchange Chromatography 10 68
10.8 Immunoaffinity Chromatography IO.73
Basic Protocol: Isolation of Soluble or Membrane Bound Antigens 10 73
Alternate Protocol 1: Batch Purification of Antigens 10 76
Alternate Protocol 2: Low pH Elution of Antigens 10 76
10.9 Metal Chelate Affinity Chromatography 10 76
Basic Protocol: Native MCAC for Purification of Soluble
Histidine Tail Fusion Proteins 10 77
Alternate Protocol 1: Denaturing MCAC for Purification of Insoluble Histidine Tail Fusion Proteins 10 79
Alternate Protocol 2: Solid Phase Renaturation of MCAC Purifie Proteins 10 81
Support Protocol: NTA Resin Regeneration 10 81
10.10 Reversed Phase Isolation of Peptides 10 82
Basic Protocol 1: Reversed Phase Peptide Separation at the 5 to 500 pmol Level 10 82
Basic Protocol 2: Reversed Phase Peptide Separation at 5 pmol 10 83
Support Protocol: Capillary HPLC System Assembly 10 84
10.11 Purification of Recombinant Proteins by Epitope Tagging 10 86
Basic Protocol 1: Immunoprecipitation of Epitope Tagged Recombinant Proteins 10 86
10.12 Immunoprecipitation 10 88
Basic Protocol: Immunoprecipitation of Radiolabeled Antigen with Antibody Sepharose 10 88
Support Protocol: Preparation of Antibody Sepharose 10 90
Alternate Protocol 1: Immunoprecipitation of Radiolabeled Antigen
with Anti Ig Serum ,q qj
Alternate Protocol 2: Immunoprecipitation of Radiolabeled Antigen with Anti Ig Sepharose,
Protein A or G Sepharose, or S. aureus Cells 10 92
Alternate Protocol 3: Immunoprecipitation Using More Strongly
Dissociating Lysis and Wash Buffers IQ.93
Alternate Protocol 4: Immunoprecipitation of Unlabeled Antigen
with Antibody Sepharose 10 p3
10.13 Synthesizing Proteins In Vitro by Transcription and
Translation of Cloned Genes 10 o4
Short Protocols in Molecular Biology Page xii ~~ ~~ ¦— j—
10.14 Metabolic Labeling with Amino Acids 10 96
Safety Precautions for Working with S Labeled Compounds 10 96
Basic Protocol: Pulse Labeling of Cells in Suspension with [ SJMethionine 10 96
Alternate Protocol 1: Pulse Labeling of Adherent Cells with [ S]Methionine 10 97
Alternate Protocol 2: Pulse Chase Labeling of Cells with f S]Methionine 10 98
Alternate Protocol 3: Long Term Labeling of Cells with [ S]Methionine 10 99
Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids 10 10C
Support Protocol: TCA Precipitation to Determine Label Incorporation 10 101
10.15 Isolation of Proteins for Microsequence Analysis 10 102
Basic Protocol 1: Determination of Amino Acid Sequence of Samples on
PVDF Membranes 10 102
Support Protocol: Preparation of Protein Samples for SDS PAGE 10 104
Basic Protocol 2: Determination of Internal Amino Acid Sequence from
N Terminally Blocked Proteins 10 104
11 IMMUNOLOGY
INTRODUCTION 11 1
11.1 Conjugation of Enzymes to Antibodies 11 3
Basic Protocol: Conjugation of HRPO to Antibodies 11 3
Alternate Protocol: Conjugation of Alkaline Phosphatase to Antibodies 11 4
11.2 Enzyme Linked Immunosorbent Assay (ELISA) 11 4
Basic Protocol: Indirect ELISA to Detect Specific Antibodies 11 4
Alternate Protocol 1: Direct Competitive ELISA to Detect Soluble
Antigens 11 6
Alternate Protocol 2: Antibody Sandwich ELISA to Detect Soluble
Antigens 11 8
Alternate Protocol 3: Double Antibody Sandwich ELISA to Detect
Specific Antibodies 11 9
Alternate Protocol 4: Direct Cellular ELISA to Detect Cell Surface
Antigens 11 11
Alternate Protocol 5: Indirect Cellular ELISA to Detect Antibodies
Specific for Surface Antigens 11 12
Support Protocol 1: Criss Cross Serial Dilution Analysis to Determine
Optimal Reagent Concentrations 11 13
Support Protocol 2: Preparation of Bacterial Cell Lysate Antigens 11 14
11.3 Production of Monoclonal Antibody Supernatant and Ascites Fluids 11 15
Basic Protocol 1: Production of a Monoclonal Antibody Supernatant 11 16
Alternate Protocol 1: Large Scale Production of Monoclonal Antibody Supernatant 11 16
Alternate Protocol 2: Large Scale Production of Hybridomas or Cell Lines 11 17
Basic Protocol 2: Production of Ascites Fluid Containing Monoclonal Antibody 11 18
11.4 Purification of Monoclonal Antibodies 11 20
Basic Protocol: Purification Using Protein A Sepharose 11 20
Alternate Protocol 1: Alternative Buffer System for Protein
A Sepharose 11 20
Alternate Protocol 2: Purification by Antigen Sepharose and Anti mouse Ig Sepharose 11 21
11.5 Production of Polyclonal Antisera in Rabbits 11 22
Basic Protocol: Intramuscular Immunization 11 22
Alternate Protocol 1: Intradermal Immunization 11 24
Alternate Protocol 2: Subcutaneous Immunization 11 24
Alternate Protocol 3: Bleeding from the Ear Artery 11 24
11.6 Purification of IgG Antibodies from Antiserum,
Ascites Fluid, or Hybridoma Supernatant 11 25
Basic Protocol: Precipitation of IgG with Ammonium Sulfate 11 25
Alternate 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 Bioloi
^w. rrwi rc .
11.8 Production of Antipeptide Antibodies 11 28
Basic Protocol: Chemical Coupling of Synthetic Peptide to
Carrier Protein Using MBS 11 29
Alternate Protocol: Chemical Coupling of Synthetic Peptide to
Carrier Protein Using Glutaraldehyde 11 29
12 DNA PROTEIN INTERACTIONS
INTRODUCTION 12 1
12.1 Preparation of Nuclear and Cytoplasmic Extracts from
Mammalian Cells 12 3
Basic Protocol: Preparation of Nuclear Extracts 12 3
Support Protocol 1: Optimization of Nuclear Extraction 12 5
Support Protocol 2: Preparation of the Cytoplasmic (S 100) Fraction 12 6
12.2 Mobility Shift DNA Binding Assay Using Gel Electrophoresis 12 6
Basic Protocol: Mobility Shift Assay 12 7
Alternate Protocol 1: Competition Mobility Shift Assay 12 9
Alternate Protocol 2: Antibody Supershift Assay 12 10
Alternate Protocol 3: Multicomponent Mobility Shift Assays 12 10
12.3 Methylation and Uracil Interference Assays for Analysis of
Protein DNA Interactions 12 11
Basic Protocol 1: Methylation Interference Assay 12 11
Basic Protocol 2: Uracil Interference Assay 12 13
12.4 DNase I Footprint Analysis of Protein DNA Binding 12 15
Basic Protocol: DNase I Footprint Titration 12 15
Support Protocol: Quantitation of Protein Binding Equilibria by
Densitometric and Numerical Analyses 12 18
Alternate Protocol: DNase Footprinting in Crude Fractions 12 20
12.5 UV Crosslinking of Proteins to Nucleic Acids 12 21
Basic Protocol: UV Crosslinking Using a BrdU Substituted Probe 12 21
Alternate Protocol 1: UV Crosslinking Using a Non BrdU Substituted Probe 12 24
Alternate Protocol 2: UV Crosslinking In Situ 12 24
12.6 Purification of DNA Binding Proteins Using Biotin/Streptavidin
Affinity Systems 12 25
Basic Protocol 12 25
Alternate Protocol 1: Purification Using a Microcolumn 12 27
Alternate Protocol 2: Purification Using Streptavidin Agarose 12 28
12.7 Detection, Purification, and Characterization of cDNA Clones
Encoding DNA Binding Proteins 12 28
Basic Protocol: Screening a A gtl 1 Expression Library with
Recognition Site DNA 12 28
Alternate Protocol: Denaturation/Renaturation Cycling of Dried Filters 12 30
Support 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 Synthesized
In Vitro from Cloned Genes 12 32
12.9 Purification of Sequence Specific DNA Binding Proteins by
Affinity Chromatography 12 33
Basic Protocol 1: Preparation of DNA Affinity Resin 13.33
Alternate Protocol: Coupling the DNA to Commercially Available CNBr Activated Sepharose 12 36
Support Protocol 1: Purification of Oligonucleotides by Preparative Gel Electrophoresis 12 41
Basic Protocol 2: DNA Affinity Chromatography 12.38
Support Protocol 2: Selection and Preparation of Nonspecific Competitor DNA 12 40
Short Protocols in Molecular Biology
12.10 Determination of Protein DNA Sequence Specificity by PCR Assisted Binding Site Selection 12 41
Basic Protocol 12 41
Support Protocol: Isolation and Analysis of Bound Oligonucleotides
from Mobility Shift Gels 12 45
13 SACCHAROMYCES CEREYISIAE
INTRODUCTION 13 1
13.1 Preparation of Yeast Media 13 2
Liquid Media 13 3
Solid Media 13 5
Strain Storage and Revival 13 7
Basic Protocol: Preparation and Inoculation of Frozen Stocks 13 7
13.2 Growth and Manipulation of Yeast 13 7
Basic Protocol 1: Growth in Liquid Media 13 8
Basic Protocol 2: Growth on Solid Media 13 8
Basic Protocol 3: Determination of Cell Density 13 8
Basic Protocol 4: Determination of Phenotype by Replica Plating 13 9
Basic Protocol 5: Diploid Construction 13 9
Basic Protocol 6: Sporulation of Diploid Cells 13 9
Basic Protocol 7: Preparation and Dissection of Tetrads 13 10
Support Protocol: Preparation of Dissecting Needles 13 12
Alternate Protocol: Random Spore Analysis 13 13
13.3 Mutagenesis of Yeast Cells 13 14
Basic Protocol: Mutagenesis Using Ethyl Methanesulfonate 13 14
Alternate Protocol: Mutagenesis Using UV Irradiation 13 16
13.4 Yeast Cloning Vectors and Genes 13 16
Plasmid Nomenclature 13 17
Maps of Selected Plasmids and Genes 13 21
13.5 Yeast Vectors for Expression of Cloned Genes 13 26
13.6 Yeast Vectors and Expression Assays 13 28
Basic Protocol 1: LacZ Fusion Vectors for Studying Gene Regulation 13 28
Basic Protocol 2: Assay for (3 galactosidase in Liquid Cultures 13 29
Alternate Protocol: Screening for p Galactosidase Expressing Yeast Colonies Using a
Filter Lift Assay 13 30
13.7 Introduction of DNA into Yeast Cells 13 31
Basic Protocol: Transformation Using Lithium Acetate 13 31
Alternate Protocol 1: Spheroplast Transformation 13 32
Alternate Protocol 2: Transformation by Electroporation 13 34
Support Protocol: Preparation of Single Stranded High Molecular Weight Carrier DNA 13 35
13.8 Cloning Yeast Genes by Complementation 13 36
13.9 Segregation of Plasmids from Yeast Cells 13 38
Basic Protocol 1 13 38
Basic Protocol 2: Plasmid Gap Repair 13 39
Basic Protocol 3: Plasmid Shuffling 13 39
13.10 Manipulation of Cloned Yeast DNA 13 41
Basic Protocol 1: Integrative Transformation 13 41
Gene Replacement Techniques 13 42
Basic Protocol 2: Integrative Disruption 13 42
Basic Protocol 3: One Step Gene Disruption 13 42
Alternate Protocol 1: PCR Mediated One Step Gene Disruption 13 43
Basic Protocol 4: Transplacement 13 44
Basic Protocol 5: Creating Modified Genes by One Step Integrative Replacement 13 45
Alternate Protocol 2: Creating Modified Genes by Transplacement 13 47
Basic Protocol 6: Creation of Conditional Alleles by Copper Inducible Double Shutoff Procedure 13 47
13.11 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 52
Alternate Protocol 2: Preparation of Poly(A)+ RNA 13 53
13.13 Preparation of Protein Extracts from Yeast 13 53
Basic Protocol: Spheroplast Preparation and Lysis 13 53
Support Protocol: Nuclei Preparation by Differential Centrifugation 13 55
Alternate Protocol 1: Cell Disruption Using Glass Beads 13 55
Alternate 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, and
Single Cells 14 2
Basic Protocol: Paraformaldehyde Fixation and Paraffin Wax
Embedding of Tissues and Embryos 14 2
Alternate Protocol: PFA Fixation of Suspended and Cultured Cells 14 3
Support Protocol 1: Perfusion of Adult Mice 14 4
Support Protocol 2: Sectioning Samples in Wax Blocks 14 5
Support Protocol 3: Preparation of Coated Slides 14 6
14.2 Cryosectioning 14_7
Basic Protocol: Specimen Preparation and Sectioning 14 7
Support Protocol 1: Fixation of Cryosections for In Situ Hybridization 14 9
Support Protocol 2: Tissue Fixation and Sucrose Infusion 14 11
14.3 In Situ Hybridization to Cellular RNA 14.11
Basic Protocol: Hybridization Using Paraffin Sections or Cells 14 11
Alternate Protocol: Hybridization Using Cryosections 14 15
Support Protocol 1: Synthesis of S Labeled Riboprobes 14 16
Support Protocol 2: Synthesis of S Labeled Double Stranded
DNA Probes 14 17
14.4 Detection of Hybridized Probe 14 17
Basic Protocol 1: Film Autoradiography 14 18
Basic Protocol 2: Emulsion Autoradiography 14 18
Support Protocol: Preparation of Diluted Emulsion for Autoradiography 14 19
14.5 Counterstaining and Mounting of Autoradiographed In Situ
Hybridization Slides 14 19
Basic Protocol: Giemsa Staining 14 19
Alternate Protocol 1: Hematoxylin/Eosin Staining 14 21
Alternate Protocol 2: Toluidine Blue Staining 14 21
Alternate Protocol 3: Hoechst Staining 14 22
14.6 Immunohistochemistry 14 22
Basic Protocol 1: Immunofluorescent Labeling of Cells Grown as
Monolayers 14 23
Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells 14 23
Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections 14 24
Alternate Protocol 2: Immunofluorescent Labeling Using Streptavidin
Biotin Conjugates 14 25
Alternate Protocol 3: Immunogold Labeling of Tissue Sections 14 26
Alternate Protocol 4: Immunoperoxidase Labeling of Tissue Sections 14 26
Alternate Protocol 5: Immunofluorescent Double Labeling of Tissue
Sections 14 28
Short Protocols in Molecular Biolom
14.7 In Situ Hybridization and Detection Using Nonisotopic Probes 14 29
Basic Protocol: Fluorescence In Situ Hybridization 14 29
Amplification of Hybridization Signals 14 31
Support Protocol 1: Amplification of Biotinylated Signals 14 31
Support Protocol 2: Amplification of Signals from Digoxigenin
Labeled Probes 14 32
Enzymatic Detection of Nonisotopically Labeled Probes 14 33
Alternate Protocol 1: Enzymatic Detection Using Horseradish Peroxidase 14 33
Alternate Protocol 2: Enzymatic Detection Using Alkaline Phosphatase 14 34
14.8 In Situ Polymerase Chain Reaction and Hybridization to Detect
Low Abundance Nucleic Acids Targets 14 35
Strategic Planning 14 35
Basic Protocol 1: In Situ PCR (ISPCR) Amplification of DNA and
RNA Targets with In Situ Reverse Transcription for RNA 14 38
Alternate Protocol: One Step Reverse Transcription and Amplification 14 42
Basic Protocol 2: Hybridization and Detection of ISPCR Amplified
Target Material 14 43
Support Protocol 1: Preparation of AES Subbed Slides 14 45
Support Protocol 2: Preparation of Specimens on Slides for ISPCR 14 45
Support 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 Procedures
and Optimization 15 3
15.2 Direct DNA Sequencing of PCR Products 15 8
Basic Protocol 1: Dideoxy Sequencing of Single Stranded Products Generated by Asymmetric PCR 15 8
Alternate Protocol 1: Generating Single Stranded Template for Dideoxy Sequencing by
Single Primer Reamplification 15 9
Alternate Protocol 2: Preparing Double Stranded PCR Products for Dideoxy Sequencing 15 10
Alternate Protocol 3: Dideoxy Sequencing of Single Strands Generated by k Exonuclease
Digestion of Double Stranded PCR Products 15 10
Basic Protocol 2: Labeling and Chemical Sequencing of PCR Products 15 11
Alternate Protocol 4: Genomic Sequencing of PCR Products 15 12
15.3 Quantitation of Rare DNA by PCR 15 13
15.4 Enzymatic Amplification of RNA by PCR 15 16
Basic Protocol: PCR Amplification of RNA Under Optimal Conditions 15 16
Alternate Protocol 1: Avoiding Lengthy Coprecipitation and Annealing Steps 15 17
Alternate Protocol 2: Introducing cDNA Directly into the Amplification Step 15 18
Support Protocol: Rapid Precipitation of Crude RNA 15 18
15.5 Ligation Mediated PCR for Genomic Sequencing and Footprinting 15 19
Basic Protocol: Ligation Mediated Single Sided PCR 15 19
Support Protocol 1: Preparation of Genomic DNA from Monolayer
Cells for DMS Footprinting 15 23
Support Protocol 2: Preparation of Genomic DNA from Suspension
Cells for DMS Footprinting 15 26
Support Protocol 3: Preparation of Genomic DNA for Chemical Sequencing 15 27
15.6 cDNA Amplification Using One Sided (Anchored) PCR 15 29
Basic Protocol 1: Amplification of Regions Downstream (3 ) of Known Sequence 15 29
Basic Protocol 2: Amplification of Regions Upstream (5 ) of Known Sequence 15 32
15.7 Molecular Cloning of PCR Products 15 34
Basic Protocol: Generation of T A Overhangs 15 34
Alternate Protocol 1: Generation of Half Sites 15 35
15.8 Differential Display of mRNA by PCR 15 37
16 PROTEIN EXPRESSION
INTRODUCTION 16 1
16.1 Overview of Protein Expression in E. coli
General Strategy for Gene Expression in E. coli j^ 3
Specific Expression Scenarios j°~^
Troubleshooting Gene Expression
16.2 Expression Using the T7 RNA Polymerase/Promoter System I6 5
Basic Protocol: Expression Using the Two Plasmid System 16 5
Alternate Protocol 1: Selective Labeling of Plasmid Encoded Proteins 16 7
Alternate Protocol 2: Expression by Infection with M13 Phage mGPl 2 16 8
16.3 Introduction to Expression by Fusion Protein Vectors 16 9
Solubility of the Expressed Protein 16 9
Stability of the Expressed Protein n
Cleavage of Fusion Proteins to Remove the Carrier 16 10
16.4 Enzymatic and Chemical Cleavage of Fusion Proteins N H
Basic Protocol 1: Enzymatic Cleavage of Fusion Proteins with Factor Xa 16 11
Support Protocol: Denaturing a Fusion Protein for Factor Xa Cleavage 16 12
Alternate Protocol 1: Enzymatic Cleavage of Fusion Proteins with Thrombin 16 13
Alternate Protocol 2: Enzymatic Cleavage of Matrix Bound GST Fusion Proteins 16 13
Alternate Protocol 3: Enzymatic Cleavage of Fusion Proteins with Enterokinase 16 14
Basic Protocol 2: Chemical Cleavage of Fusion Proteins Using Cyanogen Bromide 16 15
Alternate Protocol 4: Chemical Cleavage of Fusion Proteins Using Hydroxlamine 16 16
Alternate Protocol 5: Chemical Cleavage of Fusion Proteins by Hydrolysis at Low pH 16 16
16.5 Expression and Puriflcation of Maltose Binding Protein Fusions 16 17
Basic 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 20
Alternate Protocol: Purification of Fusion Proteins from the Periplasm 16 22
Support Protocol 2: Purifying the Cleaved Protein by Ion Exchange Chromatography 16 22
Support Protocol 3: Purifying the Cleaved Protein by Affinity Chromatography 16 23
16.6 Expression and Purification of Glutathione S Transferase Fusion
Proteins 16 M
16.7 Expression and Purification of Thioredoxin Fusion Proteins 16 27
Basic Protocol: Construction and Expression of a Thioredoxin Fusion Protein 16 27
Support Protocol 1: E. coli Lysis Using a French Pressure Cell 16 29
Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins 16 31
Support Protocol 3: Purification of Thioredoxin Fusion Proteins by Heat Treatment 16 32
16.8 Overview of the Baculovirus Expression System 16 33
Baculovirus Expression System 16 33
Post Translational Modification of Proteins in Insect Cells 16 35
Steps for Overproducing Proteins Using the Baculovirus Expression System 16 35
Choosing a Baculovirus Transfer Vector: Choosing a Baculovirus DNA 16 35
Choosing a Baculovirus DNA 16 38
Reagents, 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 40
Basic Protocol 2: Cotransfection of Insect Cells Using Linearized Baculoviral DNA 16 41
Alternate Protocol 1: Generation of Recombinant Baculovirus Using Wild Type Baculoviral DNA 16 43
Basic Protocol 3: Preparation of Baculovirus Stocks 16 45
Basic Protocol 4: Titering Baculovirus Stocks Using Plaque Assay 16 47
16.10 Expression and Purification of Recombinant Proteins Using the
Baculovirus System 16.48
Basic Protocol 1: Small Scale Expression for Initial Analysis 16 49
Support Protocol 1: Determining Time Course of Maximum Protein Production 16 50
Support Protocol 2: Metabolic Labeling of Recombinant Proteins 16 51
Basic Protocol 2: Large Scale Production of Recombinant Proteins 16 52
Basic Protocol 3: Purification of Recombinant Proteins Containing a Polyhistidine (6xHIS) Tag 16 53
Alternate 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 56
Transient Expression 16 56
Stable DNA Transfection 16 57
Amplification of Transfected DNA 16 59
Expression Vectors 16 59
Choice of Expression System 16 60
Troubleshooting 16 60
16.12 Transient Expression of Proteins Using COS Cells 16 60
16.13 Overview of the Vaccinia Virus Expression System 16 63
Vaccinia Replication Cycle 16 63
Effects of Vaccinia Infection 16 64
Vaccinia Vector Expression System 16 65
Steps For Expression of Genes Using Vaccinia Vectors 16 66
Safety Precautions for Using Vaccinia 16 66
16.14 Preparation of Cell Cultures and Vaccinia Virus Stocks 16 67
Basic Protocol 1: Culture of Monolayer Cells 16 68
Basic Protocol 2: Culture of Cells in Suspension 16 69
Basic Protocol 3: Preparation of a Vaccinia Virus Stock 16 70
Support Protocol 1: Titration of Vaccinia Virus Stocks by Plaque Assay 16 70
Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts 16 71
Basic Protocol 5: Preparation of an MVA Stock 16 72
Support 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 81
Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques 16 82
Basic Protocol 3: Amplification of a Plaque 16 84
Basic Protocol 4: Live Immunostaining of MVA Recombinants 16 85
Support Protocol 3: Coating Plates with Concanavalin A 16 87
16.16 Characterization of Recombinant Vaccinia Viruses and Their Products 16 87
Basic Protocol 1: Detection of Vaccinia DNA Using PCR 16 88
Basic Protocol 2: Detection of Vaccinia DNA Using Southern Blot Hybridization 16 90
Basic Protocol 3: Detection of Vaccinia DNA Using Dot Blot Hybridization 16 91
Alternate Protocol: Detection of Expressed Protein by a dot Blot Procedure 16 92
Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting 16 93
Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation 16 94
16.17 Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase
Hybrid System 16 95
Basic Protocol 1: Liposome Mediated Transfection Following Recombinant Vaccinia Virus
(VTF7 3) Infection 16 95
Basic Protocol 2: Coinfection with Two Recombinant Vaccinia Viruses 16 98
Basic Protocol 3: Infection of OST7 1 cells with a Single Virus 16 100
Basic Protocol 4: Gene Expression Using the Vote System 16 100
Support Protocol: Detection of Expressed Protein Using Pulse Labeling 16 101
16.18 Inducible Gene Expression Using an Autoregulatory, Tetracycline
Controlled System 16 102
Basic Protocol: Calcium Phosphate Mediated Stable Transfection of NIH3T3 Cells with
pTet tTAk and Tetracyline Regulated Target Plasmids 16 103
Support Protocol: Analysis of Target Gene Protein Expression 16 107
17 ANALYSIS OF PROTEIN PHOSPHORYLATION
INTRODUCTION 17 1
17.1 Overview of Protein Phosphorylation 17 1
17.2 Labeling Cultured Cells with T*i and Preparing Cell Lysates for
Immunoprecipitation 17 3
Basic Protocol: Labeling Cultured Cells with T»i and Lysis Using Mild Detergent 17 3
Alternate Protocol: Lysis of Cells by Boiling in SDS 17 5
17.3 Phosphoamino Acid Analysis 17 6
Basic Protocol: Acid Hydrolysis and Two Dimensional Electrophoretic
Analysis of Phosphoamino Acids 17 6
Alternate 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 10
Basic Protocol 1: Immunoblotting with Anti Phosphotyrosine
Antibodies and Detectiion Using [! ^JProtein A 17 10
Alternate Protocol: Detection of Bound Antibodies by Enhanced Chemiluminescence (ECL) 17 11
Basic Protocol 2: Identification of Phosphorylated Proteins by
Phosphatase Digestion 17 12
17.5 Detection of Phosphorylation by Enzymatic Techniques 17 13
Basic Protocol 1: Digestion of Phosphoproteins with Nonspecific Acid Phosphatases 17 14
Alternate Protocol 1: Digestion of Phosphoproteins with Nonspecific Alkaline Phosphatase 17 15
Basic Protocol 2: Digestion of Phosphoproteins with Protein Serine/Threonine Phosphatases 17 15
Alternate Protocol 2: Digestion of Phosphoproteins with Protein Tyrosine Phosphatases 17 16
Support Protocol: Measurement and Identification of Released T 17 16
17.6 Assays of Protein Kinases Using Exogenous Substrates 17 17
Strategic Planning 17 17
Basic Protocol 1: Assay for Cyclic Nucleotide Dependent Protein Kinases 17 19
Basic Protocol 2: Assay for Protein Kinase C Isoforms 17 20
Basic Protocol 3: Assay for Casein Kinases Using p Casein 17 20
Alternate Protocol: Assay for Casein Kinases Using a Peptide Substrate 17 21
Basic Protocol 4: Assay for Ca VCalmodulin Dependent Kinases 17 22
Basic Protocol 5: Assay for Tyrosine Kinases 17 23
Basic Protocol 6: In Gel Protein Kinase Assays 17 24
Support Protocol 1: Preparing a Cell Lysate for Kinase Assays 17 25
Support Protocol 2: TCA Precipitation to Determine Incorporation of Radioactivity 17 25
Support Protocol 3: Adsorption onto p81 Phosphocellulose Paper 17 26
17.7 Permeabilization Strategies to Study Protein Phosphorylation 17 28
Basic Protocol 1: Analysis of Protein Phosphorylation in Permeabilized Cells 17 28
Intact Cell Sample Preparation for Electrophoretic Analysis of Protein Phosphorylation 17 31
Alternate Protocol 1: Intact Cell Sample Preparation for SDS PAGE 17 31
Alternate Protocol 2: Intact Cell Sample Preparation for Isoelectric Focusing 17 32
18 SEQUENCE SIMILARITY SEARCHING USING THE BLAST
FAMILY OF PROGRAMS
18.1 Sequence Similarity Searching Using the BLAST Family of Programs 18 1
Accessing BLAST Programs and Documentation ! g. 1
Introduction to BLAST j g ^
Examples of BLAST Searches j g 6
Searching Strategies 18 19
Appendix A: BLAST Parameters j g 22
Appendix B: Sequence Identifier Syntax , g 23
19 ANALYSIS OF PROTEIN INTERACTIONS
INTRODUCTION 19 1
19.1 Interaction Trap/Two Hybrid System to Identify Interacting Proteins 19 5
Basic Protocol 1: Characterizing a Bait Protein 19 5
Basic Protocol 2: Performing an Interactor Hunt 19 13
Alternate Protocol 1: Rapid Screen for Interaction Trap Positives 19 20
Alternate Protocol 2: Performing a Hunt by Interaction Mating 19 22
Support Protocol 1: Preparation of Protein Extracts for Immunoblot Analysis 19 24
Support Protocol 2: Preparation of Sheared Salmon Sperm Carrier DNA 19 25
19.2 Affinity Purification of Proteins Binding to GST Fusion Proteins 19 26
Basic Protocol: GST Fusion Protein Affinity Purification 19 26
Support Protocol: Preparation of E. coli Extract 19 28
19.3 Phage Based Expression Cloning to Identify Interacting Proteins 19 29
Strategic Planning 19 29
Basic Protocol: Interaction cloning 19 31
APPENDICES
Al Reagents and Solutions Al 1
A2 Standard Measurements and Data A2 1
Useful Measurements and Data A2 1
Characteristics of Nucleic Acids A2 6
Radioactivity A2 12
Centrifuges and Rotors A2 15
A3 Commonly Used Techniques in Biochemistry and Molecular Biology A3 1
3 A Autoradiography A3 1
3B Silanizing Glassware A3 3
3C Dialysis and Ultrafiltration A3 4
3D Quantitation of DNA and RNA with Absorption and
Fluorescence Spectroscopy A3 10
3E Introduction of Restriction Enzyme Recognition Sequences
by Silent Mutation A3 12
3F Techniques for Mammalian Cell Tissue Culture A3 14
3G SafeUseofRadioisotopes A3 22
3H Statistics for the Molecular Biologist: Group Comparisons A3 33
A4 Selected Suppliers of Reagents and Equipment A4 1
A5 References A5 1
INDEX
Short Pnttacah in Mnltvnlnr Rinlnm,
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| any_adam_object | 1 |
| building | Verbundindex |
| bvnumber | BV012859580 |
| callnumber-first | Q - Science |
| callnumber-label | QH506 |
| callnumber-raw | QH506 |
| callnumber-search | QH506 |
| callnumber-sort | QH 3506 |
| callnumber-subject | QH - Natural History and Biology |
| classification_rvk | WC 4460 WD 4150 |
| ctrlnum | (OCoLC)833570139 (DE-599)BVBBV012859580 |
| dewey-full | 572.8/028 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.8/028 |
| dewey-search | 572.8/028 |
| dewey-sort | 3572.8 228 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| edition | 4. ed. |
| format | Book |
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| genre | 1\p (DE-588)4148875-1 Datensammlung gnd-content |
| genre_facet | Datensammlung |
| id | DE-604.BV012859580 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T18:34:58Z |
| institution | BVB |
| isbn | 047132938X |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-008750684 |
| oclc_num | 833570139 |
| open_access_boolean | |
| owner | DE-20 DE-19 DE-BY-UBM DE-Aug7 DE-29 DE-11 DE-188 |
| owner_facet | DE-20 DE-19 DE-BY-UBM DE-Aug7 DE-29 DE-11 DE-188 |
| physical | Getr. Zählung Ill., graph. Darst. |
| publishDate | 1999 |
| publishDateSearch | 1999 |
| publishDateSort | 1999 |
| publisher | Wiley |
| record_format | marc |
| spelling | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" ed. by Frederick M. Ausubel ... 4. ed. New York [u.a.] Wiley 1999 Getr. Zählung Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Molecular Biology cabt Techniques cabt Biologia molecular e macromolecular larpcal Biologie moléculaire - Manuels de laboratoire Biologie moléculaire - Technique Molecular Biology methods Laboratory Manuals Molecular biology Laboratory manuals Molecular biology Technique Biochemie (DE-588)4006777-4 gnd rswk-swf Praktikum (DE-588)4127380-1 gnd rswk-swf Molekulargenetik (DE-588)4039987-4 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Molekularbiologie (DE-588)4039983-7 gnd rswk-swf 1\p (DE-588)4148875-1 Datensammlung gnd-content Molekularbiologie (DE-588)4039983-7 s Methode (DE-588)4038971-6 s DE-604 Molekulargenetik (DE-588)4039987-4 s Biochemie (DE-588)4006777-4 s 2\p DE-604 Praktikum (DE-588)4127380-1 s 3\p DE-604 Ausubel, Frederick M. Sonstige oth HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008750684&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" Molecular Biology cabt Techniques cabt Biologia molecular e macromolecular larpcal Biologie moléculaire - Manuels de laboratoire Biologie moléculaire - Technique Molecular Biology methods Laboratory Manuals Molecular biology Laboratory manuals Molecular biology Technique Biochemie (DE-588)4006777-4 gnd Praktikum (DE-588)4127380-1 gnd Molekulargenetik (DE-588)4039987-4 gnd Methode (DE-588)4038971-6 gnd Molekularbiologie (DE-588)4039983-7 gnd |
| subject_GND | (DE-588)4006777-4 (DE-588)4127380-1 (DE-588)4039987-4 (DE-588)4038971-6 (DE-588)4039983-7 (DE-588)4148875-1 |
| title | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" |
| title_auth | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" |
| title_exact_search | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" |
| title_full | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" ed. by Frederick M. Ausubel ... |
| title_fullStr | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" ed. by Frederick M. Ausubel ... |
| title_full_unstemmed | Short protocols in molecular biology a compendium of methods from "Current protocols in molecular biology" ed. by Frederick M. Ausubel ... |
| title_short | Short protocols in molecular biology |
| title_sort | short protocols in molecular biology a compendium of methods from current protocols in molecular biology |
| title_sub | a compendium of methods from "Current protocols in molecular biology" |
| topic | Molecular Biology cabt Techniques cabt Biologia molecular e macromolecular larpcal Biologie moléculaire - Manuels de laboratoire Biologie moléculaire - Technique Molecular Biology methods Laboratory Manuals Molecular biology Laboratory manuals Molecular biology Technique Biochemie (DE-588)4006777-4 gnd Praktikum (DE-588)4127380-1 gnd Molekulargenetik (DE-588)4039987-4 gnd Methode (DE-588)4038971-6 gnd Molekularbiologie (DE-588)4039983-7 gnd |
| topic_facet | Molecular Biology Techniques Biologia molecular e macromolecular Biologie moléculaire - Manuels de laboratoire Biologie moléculaire - Technique Molecular Biology methods Laboratory Manuals Molecular biology Laboratory manuals Molecular biology Technique Biochemie Praktikum Molekulargenetik Methode Molekularbiologie Datensammlung |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008750684&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT ausubelfrederickm shortprotocolsinmolecularbiologyacompendiumofmethodsfromcurrentprotocolsinmolecularbiology |