Sambrook J. Molecular cloning: a laboratory manual. Vol.2 / J.Sambrook, D.W.Russell. - 3rd ed. - New York: Cold Spring Harbor Laboratory, 2001. - xxvii, P.8.1-14.53 (various pagings): ill. - Incl. bibl. ref. and index. - ISBN 978-087969577-4  Место хранения:   040 | Институт биофизики СО РАН | Красноярск | Библиотека

Оглавление / Contents

Preface ....................................................... xxi Acknowledgments ............................................. xxiii Cold Spring Harbor Protocols Online ........................... xxv Quotation Credits ........................................... xxvii Volume 2 Chapter 8 In Vitro Amplification of DNA by the Polymerase Chain Reaction ...................................................... 8.1 INTRODUCTION PROTOCOLS 1 The Basic Polymerase Chain Reaction ....................... 8.18 2 Purification of PCR Products in Preparation for Cloning ... 8.25 3 Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration .......................... 8.27 Introduction to Cloning PCR Products (Protocols 4-7) ...... 8.30 4 Blunt-end Cloning of PCR Products ......................... 8.32 5 Cloning PCR Products into T Vectors ....................... 8.35 6 Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA .............................. 8.37 7 Genetic Engineering with PCR .............................. 8.42 8 Amplification of cDNA Generated by Reverse Transcription of mRNA (RT-PCR) .......................................... 8.46 9 Rapid Amplification of 5' cDNA Ends (5'-RACE) ............. 8.54 10 Rapid Amplification of 3' cDNA Ends (3'-RACE) ............. 8.61 11 Mixed Oligonucleotide-primed Amplification of cDNA (MOPAC) ................................................... 8.66 12 Rapid Characterization of DNAs Cloned in Prokaryotic Vectors ................................................... 8.72 · Additional Protocol: Screening Yeast Colonies by PCR .... 8.75 ·Additional Protocol: Screening Bacteriophage λ Libraries ................................................ 8.76 13 Long PCR .................................................. 8.77 14 Inverse PCR ............................................... 8.81 15 Quantitative PCR .......................................... 8.86 16 Differential Display-PCR (DD-PCR) ......................... 8.96 INFORMATION PANELS Multiplex PCR ............................................... 8.107 Taq DNA Polymerase .......................................... 8.108 Hot Start PCR ............................................... 8.110 Ribonuclease H .............................................. 8.111 Terminal Transferase ........................................ 8.111 Touchdown PCR ............................................... 8.112 Use of Inosine in Degenerate Pools of Oligonucleotides Used for PCR ................................................ 8.113 Universal Primers ........................................... 8.113 Chapter 9 Preparation of Radiolabeled DNA and RNA Probes ................ 9.1 INTRODUCTION PROTOCOLS 1 Random Priming: Radiolabeling of Purified DNA Fragments by Extension of Random Oligonucleotides .................... 9.4 2 Random Priming: Radiolabeling of DNA by Extension of Random Oligonucleotides in the Presence of Melted Agarose .................................................... 9.9 ·Nick Translation: An Historical Note ..................... 9.12 3 Radiolabeling of DNA Probes by the Polymerase Chain Reaction .................................................. 9.14 ·Additional Protocol: Asymmetric Probes ................... 9.18 4 Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates ................... 9.19 5 Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates ................... 9.25 6 Synthesis of Single-stranded RNA Probes by In Vitro Transcription ............................................. 9.29 ·Additional Protocol: Using PCR to Add Promoters for Bacteriophage-encoded RNA Polymerases to Fragments of DNA ...................................................... 9.36 7 Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers ................................... 9.38 8 Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer ..................................... 9.41 9 Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension ................................. 9.46 10 Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment off. coli DNA Polymerase I ................ 9.51 11 Labeling 3' Termini of Double-stranded DNA with Bacteriophage T4 DNA Polymerase ........................... 9.57 12 End Labeling Protruding 3' Termini of Double-stranded DNA with [α-32P]Cordycepin 5' Triphosphate or [α-32P]dideoxy ATP ........................................ 9.60 13 Dephosphorylation of DNA Fragments with Alkaline Phosphatase ............................................... 9.62 14 Phosphorylation of DNA Molecules with Protruding 5'- Hydroxyl Termini .......................................... 9.66 15 Phosphorylation of DNA Molecules with Dephosphorylated Blunt Ends or Recessed 5' Termini ......................... 9.70 16 Phosphorylation of DNA Molecules with Protruding 5' Termini by the Exchange Reaction .......................... 9.73 INFORMATION PANELS Nonradioactive Labeling of Nucleic Acids ..................... 9.76 E. coli DNA Polymerase I and the Klenow Fragment ............. 9.82 In Vitro Transcription Systems ............................... 9.87 Isolating Differentially Expressed cDNAs by Differential Screening and Cloning ........................................ 9.89 Alkaline Phosphatase ......................................... 9.92 Chapter 10 Working with Synthetic Oligonucleotide Probes ................ 10.1 INTRODUCTION PROTOCOLS 1 Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis ....................... 10.11 2 Phosphorylating the 5'Termini of Oligonucleotides ........ 10.17 3 Purification of Radiolabeled Oligonucleotides by Precipitation with Ethanol ............................... 10.20 4 Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide ............... 10.22 5 Purification of Radiolabeled Oligonucleotides by Size- exclusion Chromatography ................................. 10.25 6 Purification of Radiolabeled Oligonucleotides by Chromatography on a Sep-Pak C18 Column ................... 10.28 7 Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I ..................... 10.30 8 Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts ........................................... 10.35 9 Empirical Measurement of Melting Temperature ............. 10.38 INFORMATION PANELS Oligonucleotide Synthesis ................................... 10.42 Melting Temperatures ........................................ 10.47 Methods Used to Purify Synthetic Oligonucleotides ........... 10.48 Chapter 11 Preparation of cDNA Libraries and Gene Identification ........ 11.1 INTRODUCTION PROTOCOLS 1 Construction of cDNA Libraries ........................... 11.38 Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase ........................... 11.39 Stage 2: Second-strand Synthesis ......................... 11.44 Stage 3: Methylation of cDNA ............................. 11.49 Stage 4: Attachment of Linkers or Adaptors ............... 11.52 Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose CL-4B ......................... 11.57 Stage 6: Ligation of cDNA to Bacteriophage λ Arms ........ 11.60 ·Alternative Protocol: Ligation of cDNA into a Plasmid Vector .................................................. 11.64 ·Additional Protocol: Amplification of cDNA Libraries .... 11.65 2 Construction and Screening of Eukaryotic Expression Libraries ................................................ 11.68 Stage 1: Construction of cDNA Libraries in Eukaryotic Expression Vectors .............................. 11.69 Stage 2: Screening cDNA Libraries Constructed in Eukaryotic Expression Vectors ................... 11.75 3 Exon Trapping and Amplification .......................... 11.80 Stage 1: Construction of the Library ..................... 11.82 Stage 2: Electroporation of the Library into COS-7 Cells ........................................... 11.87 Stage 3: Harvesting the mRNA ............................. 11.89 Stage 4: Reverse Transcriptase-PCR ....................... 11.91 Stage 5: Analysis of Clones .............................. 11.96 4 Direct Selection of cDNAs with Large Genomic DNA Clones ................................................... 11.98 INFORMATION PANELS Commercial Kits for cDNA Synthesis and Library Construction ............................................... 11.109 Mo-MLV Reverse Transcriptase ............................... 11.111 Homopolymeric Tailing ...................................... 11.112 λgtl0 and λgt11 ............................................ 11.113 Constructing cDNA Libraries from Small Numbers of Cells .... 11.114 In Vitro Packaging ......................................... 11.115 COS Cells .................................................. 11.116 Biotin ..................................................... 11.117 Magnetic Beads ............................................. 11.120 Ligation-independent Cloning ............................... 11.123 Chapter 12 DNA Sequencing ............................................... 12.1 INTRODUCTION PROTOCOLS ....................................... 12.1 1 Generation of a Library of Randomly Overlapping DNA Inserts .................................................. 12.10 ·Alternative Protocol: Preparation of Small Numbers of Single-stranded DNA Templates from Bacteriophage M13 .... 12.23 ·Additional Protocol: Preparation of Dephosphorylated Blunt-ended Bacteriophage M13 Vector DNA for Shotgun Cloning ................................................. 12.24 2 Preparing Denatured Double-stranded DNA Templates for Sequencing by Dideoxy-mediated Chain Termination ......... 12.26 ·Additional Protocol: Rapid Denaturation of Double- stranded DNA ............................................ 12.30 ·Additional Protocol: Purification of Plasmid DNA from Small-scale Cultures by Precipitation with PEG .......... 12.31 3 Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase) .............. 12.32 4 Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli MAO DNA Polymerase I and Single- stranded DNA Templates ................................... 12.40 5 Dideoxy-mediated Sequencing of DNA Using Taq DNA Polymerase ............................................... 12.45 6 Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers ........................ 12.51 ·Additional Protocol: Cycle Sequencing Reactions Using PCR and Internal Labeling with [α-32P]dNTPs ............. 12.60 7 Chemical Sequencing ...................................... 12.61 ·Alternative Protocol: Rapid Maxam-Gilbert Sequencing .... 12.70 ·Additional Protocol: Preparation of End-labeled DNA for Chemical Sequencing ..................................... 12.73 8 Preparation of Denaturing Polyacrylamide Gels ............ 12.74 9 Preparation of Denaturing Polyacrylamide Gels Containing Formamide ..................................... 12.81 10 Preparation of Electrolyte Gradient Gels ................. 12.83 11 Loading and Running DNA-sequencing Gels .................. 12.85 12 Autoradiography and Reading of Sequencing Gels ........... 12.90 INFORMATION PANELS Automated DNA Sequencing ................................. 12.94 Microliter Plates ....................................... 12.100 The Klenow Fragment of E. coli DNA Polymerase I ......... 12.101 Preparation of Stock Solutions of Oligonucleotide Primers for DNA Sequencing .............................. 12.103 Sequenase ............................................... 12.104 Conventional Chain-termination Sequencing of PCR- amplified DNA ........................................... 12.106 Preparation of Stock Solutions of dNTPs and ddNTPs for DNA Sequencing .......................................... 12.107 Glycerol in DNA Sequencing Reactions .................... 12.108 Compressions in DNA Sequencing Gels ..................... 12.109 7-deaza-dGTP ............................................ 12.111 Dichlorodimethylsilane .................................. 12.112 Reading an Autoradiograph ............................... 12.113 Electrical Mobility of DNA .............................. 12.114 Chapter 13 Mutagenesis .................................................. 13.1 INTRODUCTION PROTOCOLS 1 Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA .................................... 13.11 2 Oligonucleotide-directed Mutagenesis of Single-stranded DNA ...................................................... 13.15 3 In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with Dpnl ................ 13.19 4 Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis) .............. 13.26 5 Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method ..................... 13.31 6 Site-specific Mutagenesis by Overlap Extension ........... 13.36 7 Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligonucleotides ......................................... 13.40 ·Alternative Protocol: Screening Phagemid-containing Bacterial Colonies by Hybridization to Radiolabeled Oligonucleotides ........................................ 13.47 ·Alternative Protocol: Detection of Defined Mutants by PCR ..................................................... 13.48 8 Detection of Mutations by Single-strand Conformational Polymorphism and Heteroduplex Analysis ................... 13.49 9 Generation of Sets of Nested Deletion Mutants with Exonuclease III .......................................... 13.57 10 Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease ........................... 13.62 INFORMATION PANELS BAL 31 ...................................................... 13.68 Exonuclease III ............................................. 13.72 Linker-scanning Mutagenesis ................................. 13.75 Random Mutagenesis .......................................... 13.78 Alanine-scanning Mutagenesis ................................ 13.81 Mutagenic Oligonucleotides .................................. 13.82 Selecting against Wild-type DNA in Site-directed Mutagenesis ................................................. 13.84 N6-methyladenine, Dam Methylase, and Methylation- sensitive Restriction Enzymes ............................... 13.87 Commercial Kits for Site-directed Mutagenesis ............... 13.89 Glycerol .................................................... 13.90 Mutation Detection .......................................... 13.91 Chapter 14 Screening Expression Libraries ............................... 14.1 INTRODUCTION PROTOCOLS 1 Screening Expression Libraries Constructed in Bacteriophage λ Vectors ................................... 14.4 2 Screening Expression Libraries Constructed in Plasmid Vectors .................................................. 14.14 3 Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening .......................................... 14.23 ·Alternative Protocol: Adsorbing Antibodies with Lysates of Bacteriophage-infected Cells ......................... 14.25 4 Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate ........................... 14.26 5 Removal of Cross-reactive Antibodies from Antiserum: Affinity Chromatography .................................. 14.28 6 Identifying DNA-binding Proteins in Bacteriophage λ Expression Libraries ..................................... 14.31 7 Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage λ Lysogens: Lysis of Bacterial Colonies ................................................. 14.37 8 Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage λ: Lytic Infections on Agar Plates ................................................... 14.41 9 Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage λ: Lytic Infections in Liquid Medium ................................................... 14.44 INFORMATION PANELS Plasmid and Bacteriophage λ Expression Vectors .............. 14.47 Using Antibodies in Immunological Screening ................. 14.50 INDEX 1.1 ................................................... 14.50