Preface ....................................................... xxi
Acknowledgments ............................................. xxiii
Cold Spring Harbor Protocols Online ........................... xxv
Quotation Credits ........................................... xxvii
Volume 1
Chapter 1
Plasmids and Their Usefulness in Molecular Cloning ............ 1.1
INTRODUCTION PROTOCOLS
Introduction to Preparation of Plasmid DNA by Alkaline
Lysis with SDS (Protocols 1-3) ............................... 1.31
1 Preparation of Plasmid DNA by Alkaline Lysis with SDS:
Minipreparation ........................................... 1.32
2 Preparation of Plasmid DNA by Alkaline Lysis with SDS:
Midipreparation ........................................... 1.35
3 Preparation of Plasmid DNA by Alkaline Lysis with SDS:
Maxipreparation ........................................... 1.38
Introduction to Preparation of Plasmid DNA by Boiling
Lysis (Protocols 4 and 5) ................................. 1.43
4 Preparation of Plasmid DNA by Small-scale Boiling Lysis ... 1.44
5 Preparation of Plasmid DNA by Large-scale Boiling Lysis... 1.47
6 Preparation of Plasmid DNA: Toothpick Minipreparation ..... 1.51
7 Preparation of Plasmid DNA by Lysis with SDS .............. 1.55
8 Purification of Plasmid DNA by Precipitation with
Polyethylene Glycol ....................................... 1.59
9 Purification of Plasmid DNA by Chromatography ............. 1.62
10 Purification of Closed Circular DNA by Equilibrium
Centrifugation in CsCI-Ethidium
Bromide Gradients: Continuous Gradients ................... 1.65
11 Purification of Closed Circular DNA by Equilibrium
Centrifugation in CsCI-Ethidium Bromide Gradients:
Discontinuous Gradients ................................... 1.69
12 Removal of Ethidium Bromide from DNA by Extraction with
Organic Solvents .......................................... 1.72
13 Removal of Ethidium Bromide from DNA by Ion-exchange
Chromatography ............................................ 1.75
14 Removal of Small Fragments of Nucleic Acid from
Preparations of Plasmid DNA by Centrifugation through
NaCI ...................................................... 1.78
15 Removal of Small Fragments of Nucleic Acid from
Preparations of Plasmid DNA by Chromatography through
Sephacryl S-1000 .......................................... 1.80
16 Removal of Small Fragments of Nucleic Acid from
Preparations of Plasmid DNA by Precipitation with
Lithium Chloride .......................................... 1.82
17 Directional Cloning into Plasmid Vectors .................. 1.84
18 Attaching Adaptors to Protruding Termini .................. 1.88
19 Blunt-ended Cloning into Plasmid Vectors .................. 1.90
20 Dephosphorylation of Plasmid DNA .......................... 1.93
21 Addition of Synthetic Linkers to Blunt-ended DNA .......... 1.98
22 Ligating Plasmid and Target DNAs in Low-melting-
temperature Agarose ...................................... 1.103
23 The Hanahan Method for Preparation and Transformation
of Competent E. coli: High-efficiency Transformation ..... 1.105
24 The Inoue Method for Preparation and Transformation of
Competent E. coli: "Ultra-competent" Cells ............... 1.112
25 Preparation and Transformation of Competent E. coli
using Calcium Chloride ................................... 1.116
26 Transformation of E. coli by Electroporation ............. 1.119
27 Screening Bacterial Colonies Using X-gal and IPTG:
α-Complementation ........................................ 1.123
Alternative Protocol: Direct Application of X-gal and
IPTG to Agar Plates ...................................... 1.125
28 Screening Bacterial Colonies by Hybridization: Small
Numbers .................................................. 1.126
29 "Screening Bacterial Colonies by Hybridization:
Intermediate Numbers ..................................... 1.129
Alternative Protocol: Rapid Lysis of Colonies and
Binding of DNA to Nylon Filters .......................... 1.131
30 Screening Bacterial Colonies by Hybridization: Large
Numbers .................................................. 1.132
31 Lysing Colonies and Binding of DNA to Filters ............ 1.135
32 Hybridization of Bacterial DNA on Filters ................ 1.138
INFORMATION PANELS
Chloramphenicol ............................................. 1.143
Kanamycins .................................................. 1.145
pBR322 ...................................................... 1.146
Tetracycline ................................................ 1.147
Ampicillin and Carbenicillin ................................ 1.148
X-gal ....................................................... 1.149
α-Complementation ........................................... 1.149
Ethidium Bromide ............................................ 1.150
Condensing and Crowding Reagents ............................ 1.152
Purification of Plasmid DNA by PEG Precipitation ............ 1.152
Lysozymes ................................................... 1.153
Polyethylene Glycol ......................................... 1.154
Cesium Chloride and Cesium Chloride Equilibrium Density
Gradients ................................................... 1.154
DNA Ligases ................................................. 1.157
Adaptors .................................................... 1.160
Electroporation ............................................. 1.162
Chapter 2
Bacteriophage λ and Its Vectors ............................... 2.1
INTRODUCTION PROTOCOLS
1 Plating Bacteriophage λ ................................... 2.25
Additional Protocol: Plaque-Assay of Bacteriophages That
Express β-Galactosidase ................................... 2.30
Additional Protocol: Macroplaques ......................... 2.31
2 Picking Bacteriophage A. Plaques .......................... 2.32
3 Preparing Stocks of Bacteriophage λ by Plate Lysis and
Elution ................................................... 2.34
Alternative Protocol: Preparing Stocks of Bacteriophage
λ by Plate Lysis and Scraping ............................. 2.37
4 Preparing Stocks of Bacteriophage λ by Small-scale
Liquid Culture ............................................ 2.38
5 Large-scale Growth of Bacteriophage λ: Infection at Low
Multiplicity .............................................. 2.40
Alternative Protocol: Large-scale Growth of
Bacteriophage λ: Infection at High Multiplicity ........... 2.42
6 Precipitation of Bacteriophage λ Particles from Large-
scale Lysates ............................................. 2.43
7 Assaying the DNA Content of Bacteriophage λ Stocks and
Lysates by Gel Electrophoresis ............................ 2.45
8 Purification of Bacteriophage λ Particles by Isopycnic
Centrifugation through CsCI Gradients ..................... 2.47
Alternative Protocol: Purification of Bacteriophage λ
Particles by Isopycnic Centrifugation through CsCI
Equilibration Gradients ................................... 2.51
9 Purification of Bacteriophage λ Particles by
Centrifugation through a Glycerol Step Gradient ........... 2.52
10 Purification of Bacteriophage λ Particles by Pelleting/
Centrifugation ............................................ 2.54
11 Extraction of Bacteriophage λ DNA from Large-scale
Cultures Using Proteinase К and SDS ....................... 2.56
12 Extraction of Bacteriophage λ DNA from Large-scale
Cultures Using Formamide .................................. 2.59
13 Preparation of Bacteriophage λ DNA Cleaved with a Single
Restriction Enzyme for Use as a Cloning Vector ............ 2.61
14 Preparation of Bacteriophage λ DNA Cleaved with Two
Restriction Enzymes for Use as a Cloning Vector ........... 2.64
15 Alkaline Phosphatase Treatment of Bacteriophage λ Vector
DNA ....................................................... 2.68
16 Purification of Bacteriophage λ Arms: Centrifugation
through Sucrose Density Gradients ......................... 2.71
17 Partial Digestion of Eukaryotic DNA for Use in Genomic
Libraries: Pilot Reactions ................................ 2.76
18 Partial Digestion of Eukaryotic DNA for Use in Genomic
Libraries: Preparative Reactions .......................... 2.80
19 Ligation of Bacteriophage λ Arms to Fragments of Foreign
Genomic DNA ............................................... 2.84
20 Amplification of Genomic Libraries ........................ 2.87
21 Transfer of Bacteriophage DNA from Plaques to Filters ..... 2.90
Alternative Protocol: Rapid Transfer of Plaques to
Filters ................................................... 2.95
22 Hybridization of Bacteriophage DNA on Filters ............. 2.96
23 Rapid Analysis of Bacteriophage λ Isolates:
Purification of λ DNA from Plate Lysates ................. 2.101
Additional Protocol: Removing Polysaccharides by
Precipitation with СТАВ .................................. 2.105
24 Rapid Analysis of Bacteriophage λ Isolates:
Purification of λ DNA from Liquid Cultures ............... 2.106
INFORMATION PANELS
Bacteriophages: Historical Perspective ...................... 2.109
Minimizing Damage to Large DNA Molecules .................... 2.110
In Vitro Packaging .......................................... 2.110
Chapter 3
Working with Bacteriophage M13 Vectors ........................ 3.1
INTRODUCTION PROTOCOLS
1 Plating Bacteriophage M13 ................................. 3.17
2 Growing Bacteriophage M13 in Liquid Culture ............... 3.20
3 Preparation of Double-stranded (Replicative Form)
Bacteriophage M13 DNA ..................................... 3.23
4 Preparation of Single-stranded Bacteriophage M13 DNA ...... 3.26
5 Large-scale Preparation of Single-stranded and Double-
stranded Bacteriophage M13 DNA ............................ 3.30
6 Cloning into Bacteriophage M13 Vectors .................... 3.33
7 Analysis of Recombinant Bacteriophage M13 Clones .......... 3.39
·Alternative Protocol: Screening Bacteriophage M13
Plaques by Hybridization ................................. 3.41
8 Producing Single-stranded DNA with Phagemid Vectors ....... 3.42
INFORMATION PANELS
Growth Times ................................................. 3.49
Polyethylene Glycol .......................................... 3.49
Chapter 4
Working with High-capacity Vectors ............................ 4.1
INTRODUCTION PROTOCOLS
1 Construction of Genomic DNA Libraries in Cosmid Vectors ... 4.11
2 Screening an Unamplified Cosmid Library by
Hybridization: Plating the Library onto Filters ........... 4.24
·Additional Protocol: Reducing Cross-hybridization ........ 4.27
3 Amplification and Storage of a Cosmid Library:
Amplification in Liquid Culture ........................... 4.28
4 Amplification and Storage of a Cosmid Library:
Amplification on Filters .................................. 4.31
·Alternative Protocol: Amplification on Plates ............ 4.34
5 Working with Bacteriophage P1 and Its Cloning Systems ..... 4.35
·Additional Protocol: Purification of High-molecular-
weight DNA by Drop Analysis .............................. 4.44
·Alternative Protocol: Purification of High-molecular-
weight Circular DNA by Chromatography on Qiagen Resin .... 4.45
6 Transferring P1 Clones between E. coli Hosts .............. 4.46
7 Working with Bacterial Artificial Chromosomes ............. 4.48
8 Isolation of ВАС DNA from Small-scale Cultures ............ 4.53
9 Isolation of ВАС DNA from Large-scale Cultures ............ 4.55
10 Working with Yeast Artificial Chromosomes ................. 4.58
11 Growth of S. cerevisiae and Preparation of DNA ............ 4.67
12 Small-scale Preparations of Yeast DNA ..................... 4.70
13 Analyzing Yeast Colonies by PCR ........................... 4.72
14 Isolating the Ends of Genomic DNA Fragments Cloned in
High-capacity Vectors: Vectorette Polymerase Chain
Reactions ................................................. 4.74
INFORMATION PANELS
Cre-loxP ..................................................... 4.82
Large-fragment Cloning Products and Services ................. 4.86
Chapter 5
Gel Electrophoresis of DNA and Pulsed-field Agarose Gel
Electrophoresis ............................................... 5.1
INTRODUCTION PROTOCOLS
1 Agarose Gel Electrophoresis ................................ 5.4
2 Detection of DNA in Agarose Gels .......................... 5.14
3 Recovery of DNA from Agarose Gels: Electrophoresis onto
DEAE-cellulose Membranes .................................. 5.18
4 Recovery of DNA from Agarose and Polyacrylamide Gels:
Electroelution into Dialysis Bags ......................... 5.23
5 Purification of DNA Recovered from Agarose and
Polyacrylamide Gels by Anion-exchange Chromatography ...... 5.26
6 Recovery of DNA from Low-melting-temperature Agarose
Gels: Organic Extraction .................................. 5.29
·Alternative Protocol: Recovery of DNA from Agarose Gels
Using Glass Beads ........................................ 5.32
7 Recovery of DNA from Low-melting-temperature Agarose
Gels: Enzymatic Digestion with Agarase ................... 5.33
8 Alkaline Agarose Gel Electrophoresis ...................... 5.36
·Additional Protocol: Autoradiography of Alkaline
Agarose Gels ............................................. 5.39
9 Neutral Polyacrylamide Gel Electrophoresis ................ 5.40
10 Detection of DNA in Polyacrylamide Gels by Staining ....... 5.47
11 Detection of DNA in Polyacrylamide Gels by
Autoradiography ........................................... 5.49
12 Isolation of DNA Fragments from Polyacrylamide Gels by
the Crush and Soak Method ................................. 5.51
Introduction to Pulsed-field Gel Electrophoresis
(Protocols 13-20) ......................................... 5.55
13 Preparation of DNA for Pulsed-field Gel
Electrophoresis: Isolation of DNA from Mammalian Cells
and Tissues ............................................... 5.61
14 Preparation of DNA for Pulsed-field Gel Electrophoresis:
Isolation of Intact DNA from Yeast ........................ 5.65
15 Restriction Endonuclease Digestion of DNA in Agarose
Plugs ..................................................... 5.68
16 Markers for Pulsed-field Gel Electrophoresis .............. 5.71
17 Pulsed-field Gel Electrophoresis via Transverse
Alternating Field Electrophoresis Gels .................... 5.74
·Alternative Protocol: Silver Staining PFGE Gels .......... 5.77
18 Pulsed-field Gel Electrophoresis via Contour-clamped
Homogeneous Electric Field Gels ........................... 5.79
19 Direct Retrieval of DNA Fragments from Pulsed-field
Gels ...................................................... 5.83
20 Retrieval of DNA Fragments from Pulsed-field Gels
following DNA Concentration ............................... 5.86
Chapter 6
Preparation and Analysis of Eukaryotic Genomic DNA ............ 6.1
INTRODUCTION PROTOCOLS
1 Isolation of High-molecular-weight DNA from Mammalian
Cells Using Proteinase К and Phenol ........................ 6.4
·Additional Protocol: Estimating the Concentration of DNA
by Fluorometry ........................................... 6.12
2 Isolation of High-molecular-weight DNA from Mammalian
Cells Using Formamide ..................................... 6.13
3 Isolation of DNA from Mammalian Cells by Spooling ......... 6.16
4 Isolation of DNA from Mammalian Cells Grown in 96-well
Microtiter Plates ......................................... 6.19
·Additional Protocol: Optimizing Genomic DNA Isolation
for PCR .................................................. 6.22
5 Preparation of Genomic DNA from Mouse Tails and Other
Small Samples ............................................. 6.23
·Alternative Protocol: Isolation of DNA from Mouse Tails
without Extraction by Organic Solvents ................... 6.26
·Alternative Protocol: One-tube Isolation of DNA from
Mouse Tails .............................................. 6.26
·Alternative Protocol: DNA Extraction from Paraffin
Blocks ................................................... 6.27
6 Rapid Isolation of Mammalian DNA .......................... 6.28
7 Rapid Isolation of Yeast DNA .............................. 6.31
Introduction to Southern Hybridization (Protocols 8-10) ... 6.33
8 Southern Blotting: Capillary Transfer of DNA to
Membranes ................................................. 6.39
9 Southern Blotting: Simultaneous Transfer of DNA from
a Single Agarose Gel to Two Membranes ..................... 6.47
10 Southern Hybridization of Radiolabeled Probes to
Nucleic Acids Immobilized on Membranes .................... 6.50
·Additional Protocol: Stripping Probes from Membranes ..... 6.57
·Additional Protocol: Hybridization at Low Stringency ..... 6.58
INFORMATION PANELS
Formamide and Its Uses in Molecular Cloning .................. 6.59
Spooling DNA (Historical Footnote) ........................... 6.61
Rapid Hybridization Buffers .................................. 6.61
СТАВ ......................................................... 6.62
Chapter 7
Extraction, Purification, and Analysis of mRNA from
Eukaryotic Cells .............................................. 7.1
INTRODUCTION PROTOCOLS
1 Purification of RNA from Cells and Tissues by Acid
Phenol-Guanidinium Thiocyanate-Chloroform Extraction ....... 7.4
2 A Single-step Method for the Simultaneous Preparation of
DNA, RNA, and Protein from Cells and Tissues ............... 7.9
3 Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose
Chromatography ............................................ 7.13
4 Selection of Poly(A)+ RNA by Batch Chromatography ......... 7.18
Introduction to Northern Hybridization (Protocols 5-9) .... 7.21
5 Separation of RNA According to Size: Electrophoresis of
Glyoxylated RNA through Agarose Gels ...................... 7.27
6 Separation of RNA According to Size: Electrophoresis of
RNA through Agarose Gels Containing Formaldehyde .......... 7.31
7 Transfer and Fixation of Denatured RNA to Membranes ....... 7.35
·Alternative Protocol: Capillary Transfer by Downward
Flow ..................................................... 7.41
8 Northern Hybridization .................................... 7.42
9 Dot and Slot Hybridization of Purified RNA ................ 7.46
10 Mapping RNA with Nuclease S1 .............................. 7.51
11 Ribonuclease Protection: Mapping RNA with Ribonuclease
and Radiolabeled RNA Probes ............................... 7.63
12 Analysis of RNA by Primer Extension ....................... 7.75
INFORMATION PANELS
How to Win the Battle with RNase ............................. 7.82
Inhibitors of RNases ......................................... 7.83
Diethylpyrocarbonate ......................................... 7.84
Guanidinium Salts ............................................ 7.85
Nuclease S1 .................................................. 7.86
Exonuclease VII .............................................. 7.86
Mung Bean Nuclease ........................................... 7.87
Promoter Sequences Recognized by Bacteriophage-encoded RNA
Polymerases .................................................. 7.87
Actinomycin D ................................................ 7.88
INDEX 1.1 .................................................... 7.88
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