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9780879695125

Genome Analysis: A Laboratory Manual : Cloning Systems

by
  • ISBN13:

    9780879695125

  • ISBN10:

    0879695129

  • Format: Hardcover
  • Copyright: 1999-01-01
  • Publisher: Cold Spring Harbor Laboratory Pr
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List Price: $230.00

Summary

MIT Center for Genome Research, Cambridge, MA. Third volume in a four-volume set. Lab manual describing the theoretical background, lab proto- cols, and resource materials to the study of genes and genomes. Trim size: 11.5 x 8.75 inches.

Table of Contents

Companion Volumes xv
Preface xvii
Dedication xxi
Acknowledgments xxiii
Contributors xxv
Abbreviations and Acronyms xxvii
Bacterial Cloning Systems
1(86)
I. Dunham
K. Dewar
U.-J. Kim
M.T. Ross
Genomic Libraries
2(1)
Construction of Bacterial Genomic Libraries
3(8)
Characteristics of Different Bacterial Cloning Systems
3(1)
Average Insert Size and Representation of the Genome
3(3)
DNA Source
6(1)
Amount and Quality of DNA Required
7(1)
The Bacterial Host Strain
7(4)
Plating, Handling, and Storing Libraries
11(11)
Manual Library Arraying Protocol
16(2)
Manual Replication
18(2)
Robotic Replication
20(1)
Recovery of Individual Clones
21(1)
Library Screening
22(1)
Preparation of Filters Containing Arrayed Bacterial Clone Colonies
23(9)
Generation of Filters Containing Low-density Arrays of Bacterial Clone Colonies
26(2)
Generation of Filters Containing High-density Arrays of Bacterial Clone Colonies
28(2)
Lysis of Arrayed Bacterial Clone Colonies
30(2)
Hybridization-Based Screening of Arrayed Bacterial Clones
32(9)
Hybridization of Filters Containing Arrayed Bacterial Clone Colonies
36(3)
Generation of Radiolabeled Probes from PCR Products
39(2)
PCR-Based Screening of Genomic Libraries
41(8)
Preparation of DNA Pools from Arrayed Bacterial Clone Colonies
44(3)
PCR Testing of DNA from Pools of Bacterial Clones
47(2)
Contig Assembly and Chromosome Walking
49(1)
Preparation of DNA from Clone Cultures
50(8)
Minipreparation of Cloned DNA from Individual Clone Cultures
51(2)
96-well Format Micropreparation of Clone DNA
53(2)
Large-scale Alkaline-Lysis DNA Preparation for Large-insert Clones
55(3)
Restriction Enzyme-Fingerprint Analysis of Bacterial Clones
58(13)
Restriction Enzyme Fingerprinting Using Agarose Gel Electrophoresis
61(2)
Radioactive Method for Restriction Enzyme Digest Fingerprinting Using HindIII and Sau3A1
63(2)
Preparation of Markers
65(1)
Sau3A1-cut Bacteriophage λ Markers
65(1)
HinfI-cut Bacteriophage λ Markers
66(1)
Polacrylamide Gel Preparation and Electrophoresis
67(2)
Radioactive Method for Restriction Enzyme Digest Fingerprinting Using HindIII and MspI
69(2)
Computer Software for Contig Assembly
71(1)
Chromosome Walking
72(3)
Recovering End Sequences of Bacterial Clones
75(12)
Vectorette End Rescue of Bacterial Clones
79(1)
Vectorette Library Construction
79(1)
Vectorette PCR Amplification
80(2)
Direct Sequencing of Large-insert Bacterial Clones
82(5)
Cosmids
87(116)
G.A. Evans
General Principles of Cloning in Cosmids
88(2)
Advantages and Disadvantages of Cosmid Cloning Systems
88(1)
Uses and Applications of Cosmid Libraries
88(2)
Cosmid Vectors and Library Construction
90(4)
Technical Considerations: Host Strains and Clone Instability
90(1)
Structure of Cosmid Vectors
90(2)
Outline of Genomic DNA Library Construction in Cosmid Vectors
92(2)
Cosmid Library Characterization
94(5)
The Use and Handling of Cosmid Clones and Libraries
98(1)
Preparation of A Cosmid Library
99(38)
Preparation of In Vitro Packaging Extracts
105(1)
Preparation and Verification of Packaging Strains
105(1)
Preparation of Sonicated Extract from Induced BHB2690 (Prehead Lysate [PL])
106(2)
Preparation of Frozen-Thawed Lysate of Induced BHB2688 Cells (Packaging Protein Lysate [PPL]
108(2)
Preparation of Dual cos Site Vector (sCos-1) DNA
110(1)
Purification of Dual cos Site (sCos-1) Vector DNA
110(1)
Digestion of Cloning Arms with XbaI and BamHI
111(3)
Preparation of Single cos Site Vector (pWE15) DNA
114(1)
Purification of Single cos Site Vector (pWE15) DNA
114(1)
Preparative Digestion of pWE15 Vector with BamHI
115(2)
Preparation of Genomic Insert DNA for Cloning into Dual cos Site Vectors
117(2)
Partial Digest of Genomic DNA with MboI
119(2)
Test Ligation for Vector pWE15
121(1)
Preparation of Insert DNA for Cloning into Single cos Site Vectors
122(2)
Large-volume Method for DNA Isolation from Chromosomes
124(2)
Stacked-pellet Method for DNA Isolation from Chromosomes
126(2)
Library Preparation from Flow-cytometry-sorted Chromosomes
128(2)
Ligation, In Vitro Packaging, and Plating
130(3)
Subcloning YACs into Cosmids
133(1)
Purification of YAC DNA
133(1)
Preparation and Ligation of YAC DNA with MboI when DNA Is Limiting and Irreplaceable
134(1)
Preparation and Ligation of YAC DNA with MboI
135(2)
Handling of Cosmid Libraries
137(18)
Preparation of Archive Copies of Libraries
142(2)
Amplification of Cosmid Libraries
144(1)
Preparation of Arrayed Cosmid Libraries
145(2)
Recovering Clones
147(1)
Replication of Arrayed Cosmid Libraries
148(2)
Preparation of Filter Arrays (Grids) from Arrayed Clone Libraries
150(1)
Transfer of Colonies to Filters
150(1)
Posttransfer Treatment of Filters
151(2)
High-density (Automated) Cosmid Arrays
153(2)
General Characterization of Cosmid Libraries
155(1)
Hybridization of Probes to Arrayed Cosmid Filters
156(12)
Simple Screening of Cosmid Libraries by Filter Hybridization
158(1)
Plating the Library
158(1)
Making a Replica-transfer of the Colonies on Agar
158(1)
Screening the Library
159(3)
Hybridization of YAC Probes to Cosmid Arrays
162(1)
Preparation of a YAC Clone as a Probe
162(1)
Hybridization Using a Labeled Probe
163(2)
Formamide-free Hybridization to Arrayed Filters
165(2)
PERT Suppression Hybridization
167(1)
Analysis of Selected Clones
168(20)
Manual Modified Boiling Method for the Preparation of Cosmid DNA
174(2)
Manual Alkaline-Lysis Preparation of Cosmid DNA
176(2)
Automated DNA Preparation and Restriction Enzyme Digestion Using the Biomek 1000
178(2)
Partial Digestion Mapping Using T3 and T7 RNA Polymerase Sites in sCOS and pWE Vectors
180(1)
NotI Digest
180(1)
Partial Digest of NotI-digested Cosmid DNA
181(1)
Preparation and Hybridization of Radioactive Probes Complementary to the Cosmid Ends
182(2)
cos Site Mapping Using λ Terminase
184(1)
Hybridization with cos Site Arm Probes
185(1)
Preparation of End-specific RNA Probes
186(2)
Construction of Cosmid Clone Contigs By Fingerprinting
188(15)
Restriction-enzyme-based Fingerprinting
193(1)
Digestion of Cosmid DNA and Separation of Digested Products
193(1)
Transfer of DNA to Membrane
194(1)
Labeling and Hybridization of Probe
195(2)
End-labeling Restriction Fingerprinting
197(6)
Cloning into Bacteriophage P1 Vectors
203(38)
N. Sternberg
General Principles of the Bacteriophage P1 Life Cycle
204(1)
General Principles of Bacteriophage P1 Packaging
204(1)
Bacteriophage P1 Cloning Vectors
205(1)
General Cloning Strategy
206(3)
Preparation of Insert and Vector DNA
209(13)
Purification of Genomic DNA
210(2)
Partial Digestion and Size Fractionation of Purified Genomic DNA
212(3)
Purification of pAd10sacBII vector DNA
215(4)
Preparation of pAd10sacBII Vector Arms
219(2)
Ligation of vector Arms and Insert DNA
221(1)
In Vitro Packaging
222(9)
Preparation of Pacase (Stage I) Extract for Packaging Bacteriophage P1
223(2)
Preparation of Head-Tail (Stage II) Extract for Packaging Bacteriophage P1
225(3)
In Vitro Packaging of Ligated Vector/Insert DNA
228(3)
Generating Bacteriophage P1 Colonies by Infecting Host Cells with Packaged Dna
231(3)
Generation of Bacteriophage P1 Clones
232(2)
Isolation and Analysis of Bacteriophage P1 DNA
234(7)
Isolation of Bacteriophage P1 Plasmid DNA by Alkaline Lysis
235(6)
Bacterial Artificial Chromosomes
241(56)
B. Birren
V. Mancino
H. Shizuya
Vectors for Cloning Large DNA Fragments
242(2)
Procedures in BAC Cloning
244(2)
Preparation of BAC Vector DNA
246(12)
Purification of BAC Vector DNA
249(4)
Restriction Digestion of Vector DNA and Treatment with Phosphatase
253(2)
Testing the Quality of the Linearized Vector DNA
255(2)
Testing the Quality of the Dephosphorylated Vector DNA
257(1)
Preparation of Insert DNA
258(9)
Preparation, Digestion, and Size Fractionation of Insert DNA
261(5)
Running a Second Preparative Sizing Gel
266(1)
Recovery and Handling of Large DNA
267(1)
Ligation
268(6)
Purifying Size-selected DNA for Ligation
269(2)
Ligating Size-selected DNA to Vector
271(3)
Transformation Of Cells With Large DNA By Electroporation
274(6)
Preparation of Cells Competent for Electroporation
276(2)
Electroporation of Ligated BAC Vector/Insert DNA
278(2)
Determining the Efficiency Of Ligation
280(1)
Optimizing Cloning Efficiency and Insert Size
280(2)
Manipulation and Analysis of BACs
282(8)
Isolation of BAC DNA by Alkaline Lysis
286(2)
Direct PFGE of Uncut BAC DNA for Size Estimation
288(1)
Excising BAC Inserts with NotI
289(1)
Fingerprinting BACs
290(2)
Sequencing of BAC DNA
292(1)
Shotgun Sequencing of BACs
292(1)
Modification of BACs
293(4)
Yeast Artificial Chromosomes
297(338)
E.D. Green
P. Hieter
F.A. Spencer
Overview of YAC Cloning
298(1)
Primer on Yeast Genetics
299(12)
Yeast Life Cycle
299(1)
Yeast Genome
300(1)
Yeast Genetic Nomenclature
301(1)
Yeast Growth and Propagation
302(1)
Properties and Lysis of the Yeast Cell Wall
303(1)
Yeast Genetic Markers
304(1)
Standard Yeast Vectors
304(1)
Principles of Yeast Transformation
305(1)
Homologous Recombination in Yeast
306(2)
Background on Artificial Chromosomes in Yeast
308(2)
Additional Reference Materials on Yeast Genetics
310(1)
Fundamentals of YAC Cloning
311(3)
Properties of YAC Vectors
311(1)
Properties of YAC Hosts
312(1)
Issues of Yeast Colony Color Relevant to YACs
313(1)
YAC Library Construction
314(5)
General Technical Considerations
314(2)
Preparation of YAC Vector Arms
316(3)
Purification and Subsequent Treatment of YAC Vector DNA
319(3)
Enzymatic Treatment of Purified pYAC4 DNA
320(2)
Preparation of HMW Source DNA
322(1)
Source DNA for YAC Construction
322(1)
Technical Considerations for Purifying HMW Source DNA
322(1)
Restriction Digestion of Purified HMW Source DNA
323(2)
Size Fractionation of Digested Source DNA
325(3)
Size Fractionation of Digested Source DNA by Preparative PFGE
326(2)
Ligation of YAC Vector Arms and HMW Source DNA
328(5)
Ligation of YAC Vector Arms and Agarose-embedded HMW Source DNA
329(3)
Ligation of YAC Vector Arms and HMW Source DNA in Solution
332(1)
Final Processing of Ligated Vector-Source DNA
333(3)
Agarase Treatment of Agarose-embedded, Ligated Vector-Source DNA
334(2)
Spheroplast Transformation of YAC Ligation Products
336(10)
Spheroplast Transformation
340(6)
Analysis and Processing of Primary YAC Transformants
346(2)
Arraying and Long-Term Storage of YAC Libraries
348(5)
Arraying YAC Clones for Long-term Storage
350(1)
Method 1
350(1)
Method 2
351(2)
Recombinational Cloning of Large Inserts in YACs
353(2)
Screening YAC Libraries
355(2)
Logistical Factors
355(1)
Quantitative Considerations
355(1)
Other Problems Associated with Hybridization Methods
355(2)
PCR-Based Screening of YAC Libraries
357(5)
Schemes for Constructing Pools of YAC Clones
359(3)
Purification of DNA from Pools of YAC Clones
362(4)
Preparation of Crude Yeast Cell Lysates
365(1)
PCR Analysis of YAC Pool DBA
366(1)
Confirmation of Positive YACs
367(1)
Hybridization-Based Screening of YAC Libraries
368(3)
Hybridization Analysis of YAC-containing Yeast Colonies
368(1)
Arraying and Growth of YAC Clones on Membranes
368(3)
Lysis of Yeast Colonies on Membranes
371(4)
Lysing Yeast Colonies on Membrane Filters
372(3)
Screening YAC Libraries by Hybridization of Repetitive-Element PCR Products
375(1)
Care and Handling of Isolated YAC Clones
376(2)
Storage of Isolated YAC Clones
376(1)
Shipping YAC Clones
376(2)
Overview of Analyzing Isolated YACs
378(3)
Obtaining a Colony-pure YAC Isolate
378(1)
Initial Growth and Characterization of an Isolated YAC Clone
378(3)
Purification of Genomic DNA from Yeast
381(27)
Purification of Yeast DNA in Solution by a Standard Method
384(4)
Purification of Yeast DNA in Solution by a Rapid Method
388(3)
Purification of Agarose-embedded HMW Yeast DNA Using LIDS
391(5)
Purification of Concentrated Agarose-embedded HMW Yeast DNA
396(3)
Purification of Agarose-embedded HMW Yeast DNA Using Proteinase K
399(3)
Purification of Agarose-embedded HMW Yeast DNA in Microcentrifuge Tubes
402(2)
Purification of Agarose-embedded HMW Yeast DNA in 96-well Arrays
404(4)
Analysis of YACs by PFGE
408(4)
General Steps for Routine PFGE-based Analysis of YACs
409(3)
Interpretation of PFGE Results
412(3)
YAC Size
412(1)
Number of YACs
413(1)
YAC Stability
413(2)
Establishing Long-Range Restriction Maps of YACs
415(1)
Complete Digestions and Mapping of YACs Using Infrequently Cutting Restriction Enzymes
416(6)
Complete Digestion of HMW Yeast DNA with a Rare-cutting Restriction Enzyme and PFGE Analysis
417(5)
Partial Digestion and Indirect End-Label Mapping of YACs
422(7)
Partial Digestion of HMW Yeast DNA with a Rare-cutting Restriction Enzyme and PFGE Analysis
423(6)
Isolation of YAC DNA By PFGE
429(6)
Preparative Isolation of YAC DNA by PFGE
433(2)
Use of Yeast ``Window'' Strains to Facilitate YAC Isolation
435(3)
Transfer of YACs into Yeast ``Window'' Strains by Kar1- Mating
435(3)
Strategies for YAC Contig Construction
438(3)
STS-Content Mapping
438(1)
Hybridization-based Fingerprint Analysis
439(1)
PCR-based Fingerprint Analysis
439(1)
Long-range Restriction Mapping by PFGE
440(1)
PCR-Based Analysis of YACs
441(3)
Direct PCR Analysis of YACs using Yeast Cells (Colony PCR)
443(1)
Isolation of YAC Insert ends
444(1)
Isolation of YAC Insert ends by Subcloning
445(7)
Plasmid-based Subcloning of YAC Insert Ends
448(4)
Recovery of YAC Insert Ends By Plasmid Rescue
452(3)
Plasmid Rescue Recovery of YAC Insert Ends
454(1)
Retrofitting YACs to Allow Recovery of Insert Ends by Plasmid Rescue
455(7)
Recovery of YAC Insert Ends by vector Retrofitting followed by Plasmid Rescue
459(3)
Isolation of YAC Insert ends with PCR-Based Methods
462(1)
Repeat-Vector PCR
463(4)
Recovery of YAC Insert ends by Repeat-vector PCR
465(2)
Recovery of YAC Insert ends by Bubble PCR
467(7)
Bubble-PCR-based Recovery of YAC Insert Ends
469(5)
Isolating Bacterial-Based Subclones from YACs
474(5)
Hybridization of Existing Genomic Libraries with YAC-derived Probes
474(1)
Subcloning YAC Inserts into Bacterial-based Vectors
475(3)
Analysis of YAC-derived Subclones
478(1)
Analyzing YACs for the Presence of ``Chimeric'' Inserts
479(1)
Using Somatic Hybrid Cell Lines to Detect Chimeric YACs
480(2)
Hybridization-Based Analysis of Somatic Hybrid Cell Lines To Assess Chimerism
482(4)
Assessing Chimerism by Dot-Blot Hybridization Analysis of Alu-PCR Products
483(1)
Preparation of Dot Blots Containing Alu-PCR Products from Hybrid Cell Lines
483(1)
Preparation of Alu-PCR Probes from YAC Clones
484(1)
Hybridization Analysis of Alu-PCR Products Generated from Hybrid Cell Lines and YACs
485(1)
Using Fish to Detect Chimeric YACs
486(1)
Using Genetic Mapping to Detect Chimerism
486(1)
Parallel Characterization of Overlapping YACs to Detect Chimerism
486(2)
Methods for YAC Manipulation
488(1)
Transfer of YACs between Yeast Strains
489(18)
YAC Transfer by a Genetic Cross
495(7)
YAC Transfer by Retransformation
502(2)
YAC Transfer by Kar1- Mating
504(3)
Introduction Of The kar1-Δ15 Mutation Into Yeast Strains
507(5)
Introduction of kar1-Δ15 into a ura3 Yeast Strain
509(3)
Introduction of kar1-Δ15 into a lys2 Yeast Strain
512(1)
Yeast Transformation Methods
513(5)
Lithium-acetate-based Yeast Transformation
514(2)
Electroporation-based Yeast Transformation
516(2)
Recombination-Based YAC Modification
518(1)
Integrative Modification of YACs
519(5)
Introducing a Mammalian Selectable Marker into a YAC Insert by Integrative Transformation
522(2)
YAC Fragmentation
524(6)
Fragmentation of YACs by Targeting Integration at Alu and LINE Sequences
528(2)
Two-Step Replacement of Sequences within YAC Inserts
530(6)
YAC Insert Modification by Two-step Replacement
533(3)
Constructing Larger YACs by Meiotic Recombination
536(6)
Construction of Larger YACs by YAC-YAC Recombination
538(4)
YAC Amplification
542(4)
Amplification of YACs Containing a Gal-repressible Centromere and the HSV-TK Gene
544(2)
Transfer of YACs into other Cell Types
546(21)
Introduction of YACs into Mammalian Cells by Spheroplast Fusion
548(19)
APPENDICES
1 Common Reagents
567(24)
Standard Stock Solutions
568(7)
Molarities of Concentrated Acids and Bases
575(1)
Common Laboratory Solutions
576(3)
Electrophoresis Buffers, Dyes, and Gel-loading Solutions
579(4)
Media
583(5)
Antimicrobial Agents
588(3)
2 Basic Procedures
591(26)
Quantitation of Cell Concentration
592(3)
Standard Methods Used for Isolating DNA
595(1)
Extraction of DNA Samples with Organic Chemicals
595(4)
Concentration of DNA Samples by Precipitation with Alcohols
599(3)
Quantitation of DNA
602(4)
Dialysis of DNA
606(2)
Assessing the Extent of Radiolabeling in DNA Probes by Precipitation with TCA
608(2)
Dilution and Storage of Oligonucleotides
610(1)
Storage and Shipment of Biological Samples
611(6)
3 Safety Cautions
617(8)
4 Useful Facts
625(8)
Conversion Factors
626(1)
Genome Comparisons
627(1)
Average Sizes of DNA Fragments Generated by Cleavage with Restriction Enzymes
628(1)
Single-letter Abbreviations for Amino Acids
629(1)
Genetic Code
630(1)
Codon Facts
631(1)
Isotope Information
632(1)
5 Suppliers
633(2)
Index 635

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