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Gene Biotechnology 3rd edition [Hardback]

(Esperion Therapeutics, Inc., Ann Arbor, Michigan, USA), (University of Michigan, Ann Arbor, USA), (University of Michigan , Ann Arbor, Michigan, USA), (University of Michigan, Ann Arbor, Michigan, USA)
  • Formāts: Hardback, 576 pages, height x width: 234x156 mm, weight: 929 g, 1 Tables, black and white; 140 Illustrations, black and white
  • Izdošanas datums: 18-May-2011
  • Izdevniecība: CRC Press Inc
  • ISBN-10: 1439848300
  • ISBN-13: 9781439848302
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Gene Biotechnology 3rd editionPalielināt
  • Formāts: Hardback, 576 pages, height x width: 234x156 mm, weight: 929 g, 1 Tables, black and white; 140 Illustrations, black and white
  • Izdošanas datums: 18-May-2011
  • Izdevniecība: CRC Press Inc
  • ISBN-10: 1439848300
  • ISBN-13: 9781439848302
Citas grāmatas par šo tēmu:
Permanent link: https://www.kriso.lv/db/9781439848302.html
Keywords:
Covering state-of-the-art technologies and a broad range of practical applications, the Third Edition of Gene Biotechnology presents tools that researchers and students need to understand and apply today's biotechnology techniques. Many of the currently available books in molecular biology contain only protocol recipes, failing to explain the principles and concepts behind the methods outlined or to inform the reader of possible pitfalls in the methods described. Filling these gaps, this book:











Discusses a wide variety of approaches, from very basic methods to the latest, most sophisticated technologies Contains clearly detailed, step-by-step protocols with helpful troubleshooting tips Addresses the needs of researchers in academic and commercial environments Guides graduate students in designing, implementing, and evaluating experimental projects.

Each chapter covers the principles underlying methods and techniques, and includes step-by-step descriptions of each protocol, notes, tips, and a troubleshooting guide. The book includes sections on how to write a research paper for publication in English-language journals, how to protect research discoveries and inventions via patents, and practical methods of bio-calculation.

Written by a team of internationally recognized scientists, Gene Biotechnology presents protocols as well as clear and simple explanations of the key principles and concepts behind the methods. It is a single, logically organized source for the most important new methodologies. This unique resource provides the tools to help ensure success in contemporary molecular and cellular biology research.

Recenzijas

I can recommend this book to any molecular biologist without hesitation and would not be surprised if soon it establishes itself as one of the definitive guides to laboratory practice. Annals of Botany, 2005

Preface xxvii
Authors xxix
Chapter 1 Strategies for Novel Research Projects and/or Research Grant Funding
1(16)
1.1 Introduction
1(13)
References
14(3)
Chapter 2 Rapid Isolation of Specific cDNAs or Genes by PCR
17(16)
2.1 Introduction
17(1)
2.2 Isolation of Specific Full-Length cDNAs by RT-PCR Method
17(6)
2.2.1 Isolation of RNAs
18(1)
2.2.2 Design and Synthesis of Specific Forward and Reverse Primers
18(2)
2.2.3 Amplification of cDNA of Interest by RT-PCR
20(2)
2.2.4 Purification of PCR Products by High-Speed Centrifugation of Agarose Gel Slices
22(1)
2.3 Amplification and Isolation of cDNA Ends by 5'-RACE
23(1)
2.4 Amplification and Isolation of cDNA Ends by 3'-RACE
24(1)
2.5 Isolation of Gene of Interest by PCR
24(6)
2.5.1 Isolation of Genomic DNA
24(2)
2.5.2 Partial Digestion of Genomic DNA Using Sau3AI
26(1)
2.5.2.1 Optimization of Partial Digestion of Genomic DNA with Sau3AI
26(1)
2.5.2.2 Large-Scale Preparation of Partially Digested Genomic DNA
27(1)
2.5.3 Design and Synthesis of Specific Forward and Reverse Primers
28(1)
2.5.3.1 Amplification and Isolation of Exon and Intron Sequences
28(1)
2.5.3.2 Amplification and Isolation of Promoter Sequence
29(1)
2.5.4 Amplification of Specific DNA Fragments by PCR
29(1)
2.5.5 Purification of PCR Products by Agarose Gels
30(1)
2.6 Subcloning of cDNA or Gene of Interest
30(1)
2.7 Characterization of PCR Products
30(1)
References
30(3)
Chapter 3 Construction and Screening of Subtracted and Complete Expression cDNA Libraries
33(40)
3.1 Introduction
33(3)
3.2 Construction and Screening of a Subtracted cDNA Library
36(25)
3.2.1 Isolation of Total RNAs from Cell- or Tissue-Type A and B of Interest
36(2)
3.2.1.1 Protocol A. Rapid Isolation of Total RNA by Acid Guanidinium Thiocyanate-Phenol-Chloroform Method
38(1)
3.2.1.2 Protocol B. Rapid Isolation of Total RNA Using Trizol Reagent™
39(1)
3.2.1.3 Protocol C. Measurement of RNAs
40(2)
3.2.2 Purification of mRNAs from Total RNAs
42(1)
3.2.2.1 Protocol A. Purification of Poly(A)+RNAs Using Oligo(dT)-Cellulose Column
42(2)
3.2.2.2 Protocol B. Minipurification of mRNAs Using Oligo(dT) Cellulose Resin
44(1)
3.2.3 Synthesis of First Strand cDNAs from Cell/Tissue Type A or B
45(1)
3.2.3.1 Protocol A. Synthesis of First Strand cDNAs from mRNAs
45(2)
3.2.3.2 Protocol B. TCA Assay and Calculation of cDNA Yield
47(1)
3.2.3.3 Protocol C. Analysis of cDNAs by Alkaline Agarose Gel Electrophoresis
47(2)
3.2.4 Hybridization of cDNAs from Cell/Tissue Type A or B with mRNAs from Cell/Tissue Type B or A, or Vice Versa
49(1)
3.2.5 Separation of cDNA/mRNA Hybrids from Single-Stranded cDNAs by HAP Chromatography
50(1)
3.2.6 Synthesis of Double-Stranded cDNAs from Subtracted cDNAs
50(2)
3.2.7 Ligation of cDNAs to Lambda gt10 or Appropriate Vectors
52(1)
3.2.7.1 Ligation of EcoRI Linkers/Adapters to Double-Stranded, Blunt-End cDNAs
52(4)
3.2.7.2 Restriction Enzyme Digestion of Vectors
56(1)
3.2.7.3 Ligation of cDNAs to Vectors
56(1)
3.2.8 Generation of a Subtracted cDNA Library
57(1)
3.2.8.1 In Vitro Packaging
57(1)
3.2.8.2 Titration of Packaged Phage
58(2)
3.2.8.3 Amplification of cDNA Library (Optional)
60(1)
3.2.9 Isolation of Specific cDNA from the Subtracted cDNA Library
60(1)
3.2.10 Characterization of cDNA
60(1)
3.3 Construction and Screening of a Complete Expression cDNA Library
61(10)
3.3.1 General Principles and Considerations of an Expression cDNA Library
61(1)
3.3.2 Isolation of Total RNAs and Purification of mRNAs from Cells or Tissues of Interest
61(1)
3.3.3 Synthesis of cDNAs from mRNAs
61(1)
3.3.4 Ligation of cDNAs to λgt11 Expression Vectors
61(1)
3.3.5 Generation of an Expression cDNA Library
61(2)
3.3.6 Screening of the Expression Library and Isolation of the cDNA of Interest
63(1)
3.3.6.1 Method A. Immunoscreening of Expression cDNA Library Using Specific Antibodies
63(4)
3.3.6.2 Method B. Screening a cDNA Library Using 32P-Labeled DNA as a Probe
67(1)
3.3.6.3 Method C. Screening a cDNA Library Using a Nonradioactive Probe
67(1)
3.3.6.4 Method D. Isolation of λ Phage DNAs by the Liquid Method
67(2)
3.3.7 Characterization of cDNA
69(2)
References
71(2)
Chapter 4 Subcloning of Genes or DNA Fragments
73(24)
4.1 Introduction
73(1)
4.2 Restriction Enzyme Digestion of Vector or DNA Insert for Subcloning
73(5)
4.2.1 Selection of Restriction Enzymes
73(2)
4.2.2 Selection of Cloning Vectors
75(1)
4.2.3 Protocols for Restriction Enzyme Digestion
76(2)
4.3 Purification of DNA Fragments from Agarose Gels
78(3)
4.3.1 Elution of DNA Bands by High-Speed Centrifugation of Agarose Gel Slices
78(2)
4.3.2 Elution of DNA Fragment by Melting and Thawing of Agarose Gel Slices
80(1)
4.3.3 Elution of DNA Fragment Using NA45 DEAE Membrane
81(1)
4.3.4 Elution of DNA Fragments in Agarose Gel Well
81(1)
4.4 Ligation of DNA Fragments
81(2)
4.5 Single-Step Cloning by PCR
83(2)
4.6 Transformation of Ligated DNA into Bacteria
85(3)
4.6.1 Protocol
1. Preparation of Competent Cells for Transformation
85(1)
4.6.1.1 Competent Cells for CaCl2 Transformation before Ligation
85(1)
4.6.1.2 Preparation of Competent Cells for Electroporation
86(1)
4.6.2 Protocol
2. Transformation of Cells by CaCl2 Method
86(1)
4.6.3 Protocol
3. Transformation by Electroporation
87(1)
4.7 Isolation and Purification of Plasmid DNA by Alkaline Method
88(6)
4.7.1 Protocol
1. Minipreparation of Plasmid DNA
88(3)
4.7.2 Protocol
2. Large-Scale Preparation of Plasmid DNA
91(1)
4.7.3 Protocol
3. Purification of Plasmid DNA by CsCl Gradient
92(2)
4.8 Verification of DNA Insertion by Restriction Enzyme Digestion and Agarose Gel Electrophoresis
94(1)
4.9 Verification of Insertion Site by DNA Sequencing
95(1)
References
96(1)
Chapter 5 Nonisotopic and Isotopic DNA or RNA Sequencing
97(36)
5.1 Introduction
97(2)
5.2 Nonisotopic DNA Sequencing Method
99(18)
5.2.1 Protocol
1. Preparation of DNA Templates for Sequencing
99(1)
5.2.1.1 Purification of Double-Stranded Plasmid DNA Using the Alkaline Method
99(3)
5.2.1.2 Purification of ssDNA
102(1)
5.2.2 Protocol
2. Sequencing Reactions
103(1)
5.2.2.1 Method A. Sequencing Reactions for Double-Stranded Plasmid DNA
103(4)
5.2.2.2 Method B. Sequencing Reactions for ssDNA
107(1)
5.2.3 Protocol
3. Preparation of Sequencing Gels
108(1)
5.2.3.1 Method A. Pouring the Gel Mixture Horizontally into the Sandwich
109(1)
5.2.3.2 Method B. Pouring the Gel at an Angle
110(2)
5.2.4 Protocol
4. Electrophoresis
112(2)
5.2.5 Protocol
5. Transferring of DNA from Gel onto a Nylon Membrane
114(1)
5.2.6 Protocol
6. Detection
115(1)
5.2.6.1 Method A. Chemiluminescent Detection
115(1)
5.2.6.2 Method B. Colorimetric Detection
116(1)
5.3 Isotopic DNA Sequencing Method
117(2)
5.4 Use of Formamide Gels
119(1)
5.5 Extending Sequencing Far from the Primers
120(1)
5.6 DNA Sequencing by Primer Walking
120(1)
5.7 DNA Sequencing by Unidirectional Deletions
120(4)
5.7.1 Protocol
1. Performing a Series of Deletions of the Linearized DNA with Exonuclease III and Recircularization of DNA with T4 DNA Ligase
122(2)
5.8 Direct DNA Sequencing by PCR
124(3)
5.9 RNA Sequencing
127(5)
5.9.1 Protocol
1. Annealing of Primer and RNA Template
127(1)
5.9.2 Protocol
2. Labeling Reactions
127(1)
5.9.3 Protocol
3. Termination Reaction
128(4)
References
132(1)
Chapter 6 Bioinformation Superhighway and Computer Databases of Nucleic Acids and Proteins
133(22)
6.1 Introduction
133(1)
6.2 Communication with GenBank via the Internet
133(7)
6.2.1 Submission of a Sequence to GenBank
133(2)
6.2.2 Sequence Similarity Searching Using BLAST Programs
135(1)
6.2.2.1 BLASTN
135(3)
6.2.2.2 BLASTX
138(1)
6.2.2.3 BLASTP
139(1)
6.3 Computer Analysis of DNA Sequences by the GCG Program
140(13)
6.3.1 Entry and Editing of a Sequence Using the GCG Program
140(1)
6.3.1.1 Sequence Entry
140(2)
6.3.1.2 Sequence Editing or Modification
142(1)
6.3.1.3 Review of Sequence Output
142(1)
6.3.2 Combination or Assembly of Multiple Fragments into a Single Sequence
142(1)
6.3.2.1 Generating a New Project File Using the GelStart Program
142(1)
6.3.2.2 Enter Sequences to Be Assembled into the Project File Generated in the Previous Section (e.g., FRAGMENT) Using the GelEnter Program
143(1)
6.3.2.3 Compare and Identify Overlap Points of the Entered Fragments Using the GelMerge or GelOverlap Program
144(1)
6.3.2.4 Assembly and Review of the Combined Sequence Using the GelAssemble Program
145(1)
6.3.3 Identification of Restriction Enzyme Digestion Sites, Fragment Sizes, and Potential Protein Translations of a DNA Sequence
145(1)
6.3.3.1 Exhibition of Restriction Enzymes above Both Strands of a DNA Sequence and Possible Protein Translation below the Sequence Using the Map Program
146(1)
6.3.3.2 Identification of Specific Restriction Enzyme Cutting Sites and Sizes of Fragments Using MapSort Program
147(1)
6.3.4 Comparison of Similarity between Two Sequences
148(2)
6.3.5 Translation of Nucleic Acid Sequences into Amino Acid Sequences or from an Amino Acid Sequence into a Nucleic Acid Sequence
150(1)
6.3.5.1 Translate
150(1)
6.3.5.2 BackTranslate
150(1)
6.3.6 Identification of Enzyme Digestion Sites within a Peptide or Protein
151(1)
6.3.7 Obtaining Nucleotide and Amino Acid Sequences from GenBank
152(1)
References
153(2)
Chapter 7 Characterization of DNA or Genes by Southern Blot Hybridization
155(26)
7.1 Introduction
155(1)
7.2 Principles and General Considerations
155(2)
7.3 Isolation of DNA for Analysis
157(1)
7.4 Restriction Enzyme Digestion of DNA
157(1)
7.5 Agarose Gel Electrophoresis of DNAs
157(2)
7.6 Blotting of DNAs onto Nylon Membranes
159(4)
7.6.1 Method A. Upward Capillary Transfer (6 to 12 h)
159(2)
7.6.2 Method B. Downward Capillary Transfer (1 to 1.5 h Using Alkaline Buffer or 3 h Using Neutral Buffer)
161(2)
7.7 Preparation of Probes
163(8)
7.7.1 Preparation of Nonisotopic DNA Probes
163(1)
7.7.1.1 Protocol
1. Direct Labeling of ssDNA Using the ECL Kit
163(2)
7.7.1.2 Protocol
2. Random Primer Digoxigenin Labeling of dsDNA
165(2)
7.7.1.3 Protocol
3. Nick Translation Labeling of dsDNA with Biotin-11-dUTP or Digoxigenin-11-dUTP
167(1)
7.7.2 Preparation of Isotopic DNA Probes
168(1)
7.7.2.1 Protocol
1. Nick Translation Labeling of dsDNA
168(1)
7.7.2.2 Protocol
2. DE-81 Filter-Binding Assay
168(1)
7.7.2.3 Protocol
3. Trichloroacetic Acid Precipitation
168(2)
7.7.2.4 Protocol
4. Random Primer Labeling of dsDNA
170(1)
7.7.2.5 Protocol
5. 3'-End Labeling of ssDNA (Oligonucleotides) with a Terminal Transferase
170(1)
7.8 Prehybridization and Hybridization
171(1)
7.9 Washing and/or Incubation of Antibodies
172(1)
7.9.1 Protocol A. Washing of Filters Hybridized with ECL-Labeled Probes
172(1)
7.9.2 Protocol B. Washing and Antibody Incubation of Filters Hybridized with Biotin-dUTP- or DIG-UTP-Labeled Probes
173(1)
7.9.3 Protocol C. Washing of Filters Hybridized with Isotopic Probes
173(1)
7.10 Detection of Hybridized Signal(s)
173(6)
7.10.1 Method A. Chemiluminescence Detection
173(1)
7.10.2 Method B. Colorimetric Detection of Filters Hybridized with Antibody-Conjugated Probes
174(1)
7.10.3 Method C. Detection of Signals by Autoradiography
174(5)
References
179(2)
Chapter 8 Gene Overexpression by Sense RNA in Mammalian Systems
181(28)
8.1 Introduction
181(2)
8.2 Design and Selection of Plasmid-Based Expression Vectors
183(7)
8.2.1 Constitutive Promoter Vectors
183(1)
8.2.1.1 Constitutive Promoters
183(1)
8.2.1.2 Selectable Marker Genes
184(1)
8.2.1.3 Reporter Genes
184(1)
8.2.1.4 Splicing Regions
185(1)
8.2.1.5 Kozak Sequence and Enhancer Element
185(1)
8.2.2 Inducible Promoter Vectors
185(1)
8.2.3 Retrovirus Vectors
186(4)
8.3 Preparation of Plasmid Sense cDNA Constructs
190(1)
8.4 Transient Transfection of Mammalian Cells with Sense Constructs
190(8)
8.4.1 Method A. Transfection by Calcium Phosphate Precipitation
190(3)
8.4.2 Method B. Transfection by Retrovirus Vectors
193(1)
8.4.2.1 Protocol
1. Preparation of Viral Supernatant by Transient Transfection of a Packaging Cell Line with Retrovirus Vector Constructs
193(1)
8.4.2.2 Protocol
2. Production of Stably Transfected Producer Cell Lines
194(1)
8.4.2.3 Protocol
3. Determination of Viral Titer
195(1)
8.4.2.4 Protocol
4. Amplification of Virus Stock by Serial Reinfection of Fresh Target Cells
196(1)
8.4.2.5 Protocol
5. Transfection of Cells of Interest with the High Titer Stock of Replication-Incompetent Retroviruses
197(1)
8.5 Stable Transfection of Mammalian Cells with Sense DNA Constructs
198(1)
8.5.1 Method A. Transfection by Liposomes
198(1)
8.5.2 Method B. Transfection by Electroporation
198(1)
8.5.3 Method C. Transfection by Retrovirus Vectors
199(1)
8.6 Selection of Stably Transfected Cell Lines with Appropriate Drugs
199(1)
8.7 Characterization of Stably Transfected Cell Clones
200(7)
8.7.1 Analysis of Gene Overexpression at the Protein Level by Western Blotting
201(1)
8.7.2 Examination of the Expression of Sense RNA by Northern Blotting
202(1)
8.7.3 Determination of Integration Copy Number by Southern Blot Analysis
202(1)
8.7.3.1 Isolation of Genomic DNA from Cultured Cells
202(1)
8.7.3.2 Analysis of Southern Blot Data
203(1)
8.7.4 Expression Assay of a Reporter Gene
203(1)
8.7.4.1 Activity Assay of CAT
203(1)
8.7.4.2 Luciferase Assay
204(1)
8.7.4.3 β-Galactosidase Assay
205(1)
8.7.4.4 β-Galactosidase Staining of Cells
206(1)
8.8 Generation of Transgenic Mice from Sense ES Clones
207(1)
8.9 Characterization of Transgenic Mice
207(1)
References
208(1)
Chapter 9 Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies
209(22)
9.1 Introduction
209(1)
9.2 Antisense Oligonucleotide Approaches
209(5)
9.2.1 Design and Synthesis of Antisense Oligonucleotides
209(2)
9.2.2 Treatment of Cultured Cells with Antisense Oligomers and Determination of the Optimum Dose of Oligomers by Western Blot Analysis
211(2)
9.2.3 Treatment of Cultured Cells Using an Optimum Dose of Oligomers
213(1)
9.2.4 Analysis of Inhibition of Gene Expression by Western Blotting
213(1)
9.3 Design and Selection of Plasmid-Based Expression Vectors
214(1)
9.4 Preparation of Plasmid Antisense cDNA Constructs
214(1)
9.5 Transient Transfection of Cultured Cells with Antisense Constructs
215(1)
9.6 Stable Transfection of Cultured Cells with Antisense DNA Constructs
215(3)
9.6.1 Method A. Transfection by Liposomes
215(2)
9.6.2 Method B. Transfection by Microinjection
217(1)
9.6.3 Method C. Transfection by Electroporation
218(1)
9.6.4 Method D. Transfection by Retrovirus Vectors
218(1)
9.7 Selection of Stably Transfected Cell Lines with Appropriate Drugs
218(1)
9.8 Characterization of Stably Transfected Cell Clones
218(2)
9.8.1 Analysis of Gene Underexpression at the Protein Level by Western Blotting
218(1)
9.8.2 Examination of the Expression of Antisense RNA by Northern Blotting
219(1)
9.8.3 Determination of Integration Copy Number by Southern Blot Analysis
219(1)
9.8.4 Expression Assay of Reporter Genes
220(1)
9.9 Generation of Transgenic Mice
220(8)
9.9.1 Method A. Production of Transgenic Mice from Stably Transfected ES Cells
220(1)
9.9.1.1 Selection of C57BL/6J Estrous Females
220(1)
9.9.1.2 Preparation of a Bank of Sterile Males by Vasectomy
221(1)
9.9.1.3 Pairing of Estrous Females and Sterile Males
222(1)
9.9.1.4 Preparation of Blastocyst-Stage Embryos from Pseudopregnant Mice
222(1)
9.9.1.5 Preparation of Micromanipulation Apparatus
223(1)
9.9.1.6 Injection of ES Cells into Blastocysts
224(1)
9.9.1.7 Reimplantation of the Injected Blastocysts into the Uterus of Recipient Females
225(1)
9.9.2 Method B. Production of Transgenic Mice from Oocytes
225(1)
9.9.2.1 Preparation of Oocytes
225(2)
9.9.2.2 Microinjection of DNA Constructs into Oocytes
227(1)
9.9.2.3 Reimplantation of Injected Eggs into Recipient Female Mice and Generation of Founder Mice
227(1)
9.10 Characterization of Transgenic Mice
228(1)
References
229(2)
Chapter 10 Analysis of Gene Expression at Functional Genomic Level Using Northern Blotting or PCR
231(18)
10.1 Introduction
231(1)
10.2 Principles and General Considerations
232(2)
10.3 Isolation of Total RNAs and/or Purification of mRNAs
234(1)
10.4 Electrophoresis of RNAs Using Formaldehyde Agarose Gels
234(3)
10.5 Blotting of RNAs onto Nylon Membranes by the Capillary Method
237(1)
10.6 Preparation of Isotopic or Nonisotopic DNA/RNA Probes
237(4)
10.6.1 Protocol A. Preparation of DNA Probes
237(1)
10.6.2 Protocol B. Preparation of RNA Probes by Transcription in Vitro
238(3)
10.7 Hybridization and Detection of Signals
241(1)
10.8 Analysis of mRNA Expression by a Semiquantitative PCR Approach
241(5)
References
246(3)
Chapter 11 Analysis of Gene Expression at Proteomic Level via Western Blotting
249(20)
11.1 Introduction
249(2)
11.2 Principles
251(2)
11.3 Extraction of Cellular Proteins
253(2)
11.3.1 Method A. Extraction of Total Proteins Using Lauroylsarcosine Buffer
253(2)
11.3.2 Method B. Determination of Protein Concentration Using Bradford Assay
255(1)
11.4 Analysis of Proteins by SDS-PAGE and Western Blotting
255(8)
11.4.1 Protocol
1. Preparation of SDS-Separation Gels
255(1)
11.4.2 Protocol
2. Preparation of Stacking Gels
256(1)
11.4.3 Protocol
3. Electrophoresis
257(1)
11.4.4 Protocol
4. Staining and Destaining of SDS-PAGE Using a Modified CB Method
258(1)
11.4.5 Protocol
5. Transfer of Proteins from SDS-PAGE onto a Nitrocellulose or PVDF Membrane by Electroblotting
259(1)
11.4.5.1 Method A. Semidry Electroblotting
259(1)
11.4.5.2 Method B. Liquid Transfer Using a Hoeffer TE 42 Blotting Apparatus
260(1)
11.4.6 Protocol
6. Blocking and Hybridization of Membrane Filter Using Specific Antibodies
261(1)
11.4.7 Protocol
7. Detection of Hybridized Signal(s)
262(1)
11.4.7.1 Method A. Chemiluminescence Detection for Peroxidase-Conjugated or Biotin-Labeled Antibodies
262(1)
11.4.7.2 Method B. Colorimetric Detection for Alkaline Phosphatase-Conjugated Antibodies
262(1)
11.5 Analysis of Proteins by 2-D Gel Electrophoresis
263(3)
References
266(3)
Chapter 12 Analysis of Cellular DNA or Abundance of mRNA by Radioactivity in Situ Hybridization
269(22)
12.1 Introduction
269(1)
12.2 Tissue Fixation, Embedding, Sectioning, and Mounting of Sections on Slides
270(5)
12.2.1 Preparation of Silane-Coated Glass Slides
270(1)
12.2.1.1 Method A. Silanization of Slides
270(1)
12.2.1.2 Method B. Treatment of Slides with Poly-L-Lysine
270(1)
12.2.1.3 Method C. Gelatin-Coating of Slides
270(1)
12.2.2 Preparation of Fixation Solutions
270(1)
12.2.2.1 Preparation of 4% (w/v) PFA Fixative (11)
270(1)
12.2.3 Fixation of Cultured Cells on Slides
271(1)
12.2.4 Tissue Fixation, Embedding, Sectioning, and Mounting of Sections on Slides
271(1)
12.2.4.1 Fixation
271(1)
12.2.4.2 Embedding
272(1)
12.2.4.3 Sectioning and Mounting
273(1)
12.2.5 Cryosectioning
273(1)
12.2.5.1 Preparation of Frozen Specimens
273(1)
12.2.5.2 Freeze Sectioning
274(1)
12.2.5.3 Fixation
274(1)
12.3 In Situ Hybridization and Detection Using Isotopic Probes
275(14)
12.3.1 Dewaxing of Sections
275(1)
12.3.2 Protease Digestion
276(1)
12.3.3 DNase Treatment for in Situ Hybridization of RNA
277(1)
12.3.4 RNase Treatment for in Situ Hybridization of DNA
277(1)
12.3.5 Preparation of Radioactive Probes
278(1)
12.3.5.1 Synthesis of Probes for DNA Hybridization Using Random Primer Labeling of dsDNA
278(3)
12.3.5.2 Preparation of [ 35S]UTP Riboprobe for RNA Hybridization by Transcription in Vitro Labeling
281(2)
12.3.6 In Situ Hybridization
283(2)
12.3.7 Detection of Hybridized Signals
285(4)
References
289(2)
Chapter 13 Localization of DNA or Abundance of mRNA by Fluorescence in Situ Hybridization
291(12)
13.1 Introduction
291(1)
13.2 Cell or Tissue Fixation, Tissue Embedding, Sectioning, and Mounting
291(1)
13.3 Dewaxing of Sections, Protease Digestion, and DNase or RNase Treatment of Specimens for in Situ Hybridization
292(1)
13.4 Preparation of Nonisotopic Probes Using One of the Following Methods, Depending on Specific Probe and Detection Strategies
292(4)
13.4.1 Method A. Random Primer Digoxigenin Labeling of dsDNA
292(1)
13.4.2 Method B. Nick Translation Labeling of dsDNA with Biotin-11-dUTP or Digoxigenin-11-dUTP
293(1)
13.4.3 Method C. Riboprobe or RNA Labeling
293(3)
13.5 In Situ Hybridization of Specimens
296(1)
13.6 Enzymatic Detection of Hybridized Signals Using Colorimetric Substrates NBT and BCIP
297(1)
13.7 Fluorescence Detection of Hybridized Signals
298(3)
References
301(2)
Chapter 14 In Situ PCR Hybridization of Low Copy Genes and in Situ RT-PCR Detection of Low Abundance mRNAs
303(16)
14.1 Introduction
303(1)
14.2 Detection of a Low Copy Gene by in Situ PCR Hybridization
304(8)
14.2.1 Design of Specific Primers for in Situ PCR Amplification of Target DNA Sequences
305(2)
14.2.2 Synthesis and Testing of the Designed Primers
307(1)
14.2.3 Carrying Out Fixation of Cells or Tissues, Tissue Embedding, Sectioning, and Mounting of Sections on Slides
307(1)
14.2.4 Dewaxing of Sections (if Necessary), Protease Digestion, and DNase or RNase Treatment of Specimens
308(1)
14.2.5 Preparation of Radioactive Probes for RISH or Nonradioactive Probes for FISH
308(1)
14.2.6 Performance of in Situ PCR
308(3)
14.2.7 Performance of in Situ Hybridization and Detection by RISH or by FISH
311(1)
14.3 Detection of Low Abundance mRNA by in Situ RT-PCR Hybridization
312(4)
14.3.1 Carrying Out Cell or Tissue Fixation, Tissue Embedding, Sectioning, and Mounting of Sections on Slides
312(2)
14.3.2 Dewaxing of Sections, Protease Digestion, and DNase or RNase Digestion of Specimens
314(1)
14.3.3 Performance in Situ Reverse Transcription Reaction on Sections
314(1)
14.3.4 Performance of RNase (DNase-Free) Digestion of Specimens to Eliminate Potential Mispriming between PCR Primers and mRNA Molecules
315(1)
14.3.5 Preparation of Radioactive Probes for RISH or Nonradioactive Probes for FISH
315(1)
14.3.6 Design of Specific Primers for in Situ PCR
315(1)
14.3.7 Carrying Out in Situ PCR
315(1)
14.3.8 Performance of in Situ Hybridization and Detection by RISH or by FISH
315(1)
14.4 In Situ PCR or RT-PCR Detection of DNA or mRNA by Direct Incorporation of Digoxigenin-11-dUTP or Biotin-dUTP without Hybridization
316(1)
References
317(2)
Chapter 15 Isolation and Characterization of Genes from Genomic DNA Libraries
319(22)
15.1 Introduction
319(1)
15.2 Selection of Lambda Vectors
320(1)
15.3 Isolation of High Molecular Weight of Genomic DNA
320(2)
15.4 Partial Digestion of Genomic DNA Using Sau3AI
322(2)
15.4.1 Determination of the Optimal Partial Digestion of Genomic DNA with Sau3AI
322(1)
15.4.2 Large-Scale Preparation of Partial Digestion of Genomic DNA
323(1)
15.5 Partial Fill-In of Recessed 3'-Termini of Genomic DNA Fragments
324(1)
15.6 Ligation of DNA Inserts to Vectors
325(3)
15.6.1 Small-Scale Ligation of Partially Filled-In Genomic DNA Fragments and Partially Filled-In LambdaGEM-12 Arms
325(1)
15.6.2 Packaging of the Ligated DNA
326(1)
15.6.3 Titration of Packaged Phage on Luria-Bertani Plates
326(2)
15.6.4 Large-Scale Ligation of the Partially Filled-In Vector Arms and Partially Filled-In Genomic DNA Fragments
328(1)
15.7 Screening of Genomic DNA Library and Isolation of Specific Clones
328(7)
15.7.1 Method A. Screening of a Genomic Library with α-32P-Labeled Probe
328(5)
15.7.2 Method B. Screening of a Genomic Library Using a Nonisotope Labeled Probe
333(2)
15.8 Isolation of λ Phage DNAs by the Liquid Method
335(1)
15.9 Restriction Mapping of Recombinant Bacteriophage DNA Containing the Genomic DNA Insert of Interest
336(2)
15.10 Subcloning of the Isolated Gene or DNA Fragments
338(1)
15.11 Characterization of the Isolated DNA
338(1)
References
339(2)
Chapter 16 Mouse Stem Cells as a Model Mammalian Cell Line for Gene Expression
341(10)
16.1 Introduction
341(1)
16.2 Protocol
1. Culture of ES Cells in Media Containing Leukemia Inhibitory Factor
341(2)
16.3 Protocol
2. Preparation of Mitotically Inactivated STO Cells as Feeder Layers for Growth of ES Cells
343(1)
16.4 Protocol
3. Culture of ES Cells on Fibroblast Feeder Layers
344(1)
16.5 Protocol 4: Trypsinizing, Freezing, and Thawing of Mammalian Cells
344(3)
16.5.1 Trypsinizing
344(2)
16.5.2 Freeze Back
346(1)
16.5.3 Thawing and Reculture
346(1)
16.6 Protocol
5. Cell Counting and Determination of Cell Density
347(2)
References
349(2)
Chapter 17 Strategies for Gene Double Knockout
351(30)
17.1 Introduction
351(1)
17.2 Overview and General Principles of Strategies: Gene Double-Knockout and Temporal Silence
352(2)
17.3 Manipulation of the Specific Gene to Be Targeted
354(8)
17.3.1 Protocol A. Disruption of an Exon in a Specific Gene
355(1)
17.3.2 Protocol B. Deletion of Exons of a Specific Gene
355(1)
17.3.3 Protocol C. Preparation of Isogenic DNA by PCR
356(1)
17.3.3.1 Design of Two Pairs of Primers
356(2)
17.3.3.2 Amplification of 5'- and 3'-Isogenic DNA Fragments by PCR
358(2)
17.3.3.3 Electrophoresis of PCR Products
360(2)
17.3.3.4 Purification of PCR Bands from the Agarose Gel and Ligation of Isogenic DNA Fragments with a Selectable Marker Gene
362(1)
17.4 Design and Selection of Vectors and Marker Genes for Gene Double Knockout and Temporal Silence
362(3)
17.4.1 Type A. neor Gene Vector for the First Copy of Gene Knockout
363(1)
17.4.2 Type B. hygr Gene Vector for the Second Copy of Gene Knockout
364(1)
17.4.3 Type C. hyrtr Gene Vector for Keeping the Double Knockout Gene Silent
364(1)
17.5 Preparation of DNA Constructs for a Double Knockout and a Silencer
365(2)
17.6 Transfection of ES Cells with Double Knockout and Silencer DNA Constructs
367(3)
17.6.1 Method A. Transfection by Electroporation
367(1)
17.6.2 Method B. Transfection of Cells by Lipofectin Reagents
368(2)
17.7 Selection and Characterization of Targeted ES Cell Clones
370(1)
17.7.1 Double Selections of Cells Containing a Double Knockout Gene
370(1)
17.7.2 Triple Selections of Cells Containing a Double Knockout Gene
371(1)
17.7.3 Single Selection of Cells Containing a Silent Knockout Gene
371(1)
17.8 Characterization of Targeted ES Cell Clones
371(6)
17.8.1 β-Galactosidase Staining of Cells
371(1)
17.8.2 Southern Blot Analysis
372(1)
17.8.2.1 Isolation of Genomic DNA from Cultured Cells
373(1)
17.8.2.2 Analysis of Southern Blot Data
373(2)
17.8.2.3 Western Blot Analysis
375(2)
17.9 Generation of Transgenic Offspring from Double-Knockout ES Cells
377(2)
17.9.1 Procedure
1. Selection of C57BL/6J Estrous Females
377(1)
17.9.2 Procedure
2. Preparation of a Bank of Sterile Males by Vasectomy
378(1)
17.9.3 Procedure
3. Pairing of Estrous Females and Sterile Males
378(1)
17.9.4 Procedure
4. Preparation of Blastocyst-Stage Embryos from Pseudopregnant Mice
378(1)
17.9.5 Procedure
5. Preparation of Micromanipulation Apparatus
378(1)
17.9.6 Procedure
6. Injection of ES Cells into Blastocysts
378(1)
17.9.7 Procedure
7. Reimplantation of Injected Blastocysts into the Uterus of Recipient Females
378(1)
17.10 Characterization of Mutant Mice
379(1)
References
379(2)
Chapter 18 Large-Scale Expression and Purification of Recombinant Proteins in Cultured Cells
381(34)
18.1 Introduction
381(1)
18.2 Expression and Purification of Proteins in Prokaryotic Systems
382(13)
18.2.1 Design and Selection of Bacterial Expression Vectors
382(2)
18.2.2 Cloning of cDNA Inserts into Vectors
384(2)
18.2.3 Expression of Proteins of Interest in Bacteria
386(1)
18.2.3.1 Protocol A. Transformation of E. coli BL21 (DE3) with Vector Shown in Figure 18.1 Using CaCl2 Method
386(1)
18.2.3.2 Protocol B. Induction of Protein Expression with IPTG
386(1)
18.2.3.3 Protocol C. Transformation of E. coli Strains HB101, JM109, TOP10, or DH5&alfalF' Using Vector Type Shown in Figure 18.2
387(1)
18.2.3.4 Protocol D. Induction of Protein Expression by Heat Shock
388(1)
18.2.4 Purification and Cleavage of Expressed Proteins
388(1)
18.2.4.1 Analysis of Cellular Proteins by SDS-PAGE, Dot Blot, or Western Blotting
388(1)
18.2.4.2 Extraction of Total Proteins for Purification
389(1)
18.2.4.3 Rapid Purification of Expressed Proteins with His-Tag Affinity Column Chromatography
390(2)
18.2.4.4 Protease Cleavage of His-Tag
392(3)
18.3 Overexpression of Specific Proteins in Eukaryotic Systems
395(17)
18.3.1 Cloning of a cDNA to Be Expressed into Expression Vectors
398(1)
18.3.2 Transformation of Yeast Pichia with Expression DNA Constructs
399(1)
18.3.2.1 Protocol A. Linearization of Plasmid DNA
399(1)
18.3.2.2 Protocol B. Transformation of Pichia by Electroporation
400(2)
18.3.2.3 Protocol C. Preparation of Spheroplasts for Transformation
402(3)
18.3.2.4 Protocol D. Transformation of Spheroplasts with Linearized DNA
405(2)
18.3.3 Screening and Isolation of His+Mut+ and His+Muts Phenotype
407(2)
18.3.4 Identification of Transformants Containing cDNA to Be Expressed
409(1)
18.3.5 Expression of Proteins in Transformants
410(1)
18.3.5.1 Determination of Optimum Conditions for Protein Expression
410(1)
18.3.5.2 Large-Scale Expression of Proteins
411(1)
18.3.5.3 Extraction of Total Proteins for Purification
412(1)
18.3.6 Carry Out Fusion Protein Purification and Cleavage
412(1)
References
412(3)
Chapter 19 Quantitative Analysis of Functional Genome by Real-Time RT-PCR
415(14)
19.1 Introduction
415(2)
19.2 Isolation of Total RNA from Tissues or Organs of Interest
417(1)
19.3 Isolation of Total RNA from Cultured Cells
418(1)
19.4 Preparation of RNA Pool
419(1)
19.5 Designing Primers and Probes
420(1)
19.6 Real-Time RT-PCR
421(6)
19.6.1 Assembly of Reactions
421(1)
19.6.2 Programming and Performance of Real-Time RT-PCR
422(1)
19.6.3 Analysis of Data
423(3)
19.6.4 Validation
426(1)
References
427(2)
Chapter 20 High-Throughput Analysis of Gene Expression by Cutting-Edge Technology---DNA Microarrays (Gene Chips)
429(10)
20.1 Introduction
429(1)
20.2 Isolation of Total RNA from Cells or Tissues
430(1)
20.3 Preparation of Fluorescent Probes from mRNA
431(2)
20.4 Generation of DNA Microarrays or Chips
433(1)
20.5 Prehybridization of Gene Chips or Arrays
433(1)
20.6 Hybridization
434(1)
20.7 Washing
434(1)
20.8 Scanning of Gene Chips Using GenePix 4000a Microarray Scanner
435(2)
References
437(2)
Chapter 21 Construction and Screening of Human Antibody Libraries Using Phage Display Technology
439(18)
21.1 Introduction
439(2)
21.2 Preparation of White Blood Cells (WBCs) or Lymphocytes for RNA Isolation
441(1)
21.2.1 Preparation of WBCs from Blood
441(1)
21.2.2 Preparation of Lymphocytes from Tissues
442(1)
21.3 Isolation of Total RNA from Lymphocytes
442(1)
21.4 Synthesis of cDNAs
442(4)
21.4.1 Synthesis of First Strand cDNAs from mRNAs
442(3)
21.4.2 Synthesis of Double-Stranded cDNAs from Subtracted cDNAs
445(1)
21.5 Design of Primers for Construction of Antibody V Gene Repertoire
446(3)
21.6 PCR Amplification of Heavy- and Light-Chain Variable Regions (VH, VL), Linker, and Assembly of Recombinant Single-Chain DNA via PCR
449(2)
21.7 Generation of Recombinant DNA Constructs for Expressing Recombinant Antibodies on Surface of Phage
451(1)
21.8 Expression, Biopanning, and Characterization of Phage Produces Monoclonal Antibodies on Its Tip
451(5)
21.8.1 Infection of Bacteria Carrying Recombinant DNA Constructs and Rescuing Phagemid Libraries
452(3)
21.8.2 Selection of Phage-Antibody Particles by Biopanning
455(1)
References
456(1)
Chapter 22 Down-Regulation of Gene Expression in Mammalian Systems via siRNA Technology
457(14)
22.1 Introduction
457(1)
22.2 Design of Oligonucleotides for Hairpin siRNA
458(1)
22.2.1 Selection of siRNA Target Site
458(1)
22.2.2 Selection of Oligomer Size and Orientation for siRNAs
459(1)
22.2.3 Synthesis of Hairpin siRNA-Encoding Oligonucleotides
459(1)
22.3 Preparation of Double-Stranded Oligonucleotide Fragment for Cloning
459(1)
22.4 Cloning Hairpin siRNA Oligo Insert into Appropriate Vectors
460(2)
22.5 Purification of Plasmid DNA Carrying Hairpin siRNA Insert
462(1)
22.6 Transient Transfection of Cells of Interest with Purified Plasmid DNA to Express siRNAs for Inhibiting Target mRNA
463(2)
22.7 Isolation of Total RNA from Transfected Cells
465(1)
22.8 Analysis of Inhibition of Specific mRNA via siRNAs by Northern Blotting
466(1)
22.9 Extraction of Proteins for Proteomic and/or Reporter Gene Analysis
466(1)
22.9.1 Isolation of Proteins for Western Blotting
466(1)
22.9.2 Extraction of Proteins for Reporter Activity Assays
467(1)
22.10 Analysis of siRNA-Mediated Inhibition of Gene Expression at Proteomic Level by Western Blotting
467(1)
22.11 Reporter Gene Assays of Down-Regulation of Gene Expression via siRNAs
467(1)
References
468(3)
Chapter 23 Strategies for Gene Cloning, Expression, and Identification of Protein-Protein Interaction
471(12)
23.1 Introduction
471(1)
23.2 Rapid and High-Efficiency PCR-TA Cloning without DNA Ligase
471(4)
23.2.1 Selection of Cloning Vector
472(1)
23.2.2 PCR Primer Design
473(1)
23.2.3 Performance of PCR Reactions
473(1)
23.2.4 TA Cloning
474(1)
23.2.5 Transformation of Chemically Treated E. coli One-Shot TOP10 Competent Cells
474(1)
23.2.6 Confirmation of DNA Sequences of Colonies
475(1)
23.3 Transfection of Eukaryotic Cells for Expression of Proteins of Interest
475(1)
23.3.1 Use of FuGene 6 Reagent
475(1)
23.3.2 Use of Lipofectamine 2000
476(1)
23.4 Protein Extraction
476(1)
23.5 Coimmunoprecipitation
477(1)
23.6 Analysis and Identification of Binding Proteins
477(2)
23.7 Different Strategies for Cloning, Identification, Mapping, and Characterization of Protein-Protein Interaction
479(2)
23.7.1 Use of Fluorescent Protein as a Fusion Tag and Analysis by Fluorescent Microscopy
479(1)
23.7.2 Cloning and Expression of Proteins of Interest with a Detection Minitag
479(1)
23.7.3 How to Design, Clone, and Map Binding Regions of Proteins of Interest
480(1)
23.8 Exploring the Biology or Potential Mechanisms of the Interactive Proteins
481(1)
References
481(2)
Chapter 24 Conditional Gene Knockout
483(18)
24.1 Introduction
483(1)
24.2 Generation of Cre-loxP DNA Constructs
484(4)
24.2.1 Selection of Gene or Exon(s) of Interest to Be Targeted
484(1)
24.2.1.1 Rapid Isolation of Total RNAs Using Trizol Reagent™
485(1)
24.2.1.2 Design of Primers for RT-PCR Using mRNA as Template
486(1)
24.2.1.3 Performance of RT-PCR
486(1)
24.2.2 Selection of Cloning Vector(s)
487(1)
24.2.3 Selection of Promoter for Expression of Cre
487(1)
24.2.4 Cloning
487(1)
24.3 Generation of Transgenic Mouse Line Containing loxP Cassette, Cre Cassette, or Both
488(9)
24.3.1 Method
1. Production of Transgenic Mice from Stably Transfected or Microinjected ES Cells
489(1)
24.3.1.1 Selection of C57BL/6J Estrous Females
489(1)
24.3.1.2 Preparation of a Bank of Sterile Males by Vasectomy
490(1)
24.3.1.3 Pairing of Estrous Females and Sterile Males
491(1)
24.3.1.4 Preparation of Blastocyst-Stage Embryos from Pseudopregnant Mice
491(1)
24.3.1.5 Preparation of Micromanipulation Apparatus
492(1)
24.3.1.6 Injection of Stably Transfected ES Cells into Blastocysts
493(1)
24.3.1.7 Reimplantation of Injected Blastocysts into the Uterus of Recipient Females
494(1)
24.3.2 Method
2. Production of Transgenic Mice from Oocytes Injected with DNA Constructs
494(1)
24.3.2.1 Preparation of Oocytes
494(2)
24.3.2.2 Microinjection of DNA Constructs into Oocytes
496(1)
24.3.2.3 Reimplantation of Injected Eggs into Recipient Female Mice and Generation of Founder Mice
496(1)
24.4 Generation and Characterization of Stable Transgenic Mouse Lines A and B
497(1)
24.5 Generation and Characterization of Transgenic Mouse Line C
497(2)
24.5.1 Genotypic Analysis
498(1)
24.5.2 Functional Genome Analysis
498(1)
24.5.3 Reporter Gene
499(1)
24.5.4 Immunostaining
499(1)
24.5.5 Functional and Phenotypic Analyses
499(1)
References
499(2)
Chapter 25 How to Write a Research Manuscript for Publication in an English Journal
501(10)
25.1 Introduction
501(2)
25.2 Title
503(1)
25.3 Authors and Affiliation
503(1)
25.4 Materials and Methods
504(1)
25.5 Results
505(1)
25.6 Abstract (Summary in Some Journals)
506(1)
25.7 Introduction
506(1)
25.8 Discussion
507(1)
25.9 Acknowledgments
508(1)
25.10 References
508(1)
25.11 Adjustment of Format of Manuscript
508(1)
25.12 Fine-Tuning and Submission of Manuscript
509(1)
25.13 Some Observations on to Which Journal to Submit Your Manuscript
509(1)
25.14 Waiting for Reviewers' Comments, Revision of Manuscript Based on Reviewers' Critique, and Resubmission of Manuscript
509(1)
References
510(1)
Chapter 26 How to Protect Your Discovery and Invention: Patent 101
511(10)
26.1 Introduction
511(1)
26.2 Research Discoveries vs. IPs, Invention
511(1)
26.3 IP or Invention Documentation
512(1)
26.4 Technology Disclosure
512(1)
26.5 Provisional Application for Patent
513(1)
26.6 Full-Length or Complete Patent Application
514(5)
26.6.1 Title
514(1)
26.6.2 Inventor(s)
514(1)
26.6.3 Assignee
515(1)
26.6.4 Abstract
515(1)
26.6.5 Background of Invention
515(1)
26.6.6 Summary of Invention
516(1)
26.6.7 Brief Description of Drawings and Figures
516(1)
26.6.8 Description of Illustrative Embodiments
516(2)
26.6.9 References
518(1)
26.6.10 Claims of Invention
518(1)
References
519(2)
Chapter 27 Determination of Transgene Copy Numbers and Practical Biocalculation
521(10)
27.1 Introduction
521(1)
27.2 Determination of Gene Copy Numbers in Transgenic Animals
521(3)
27.3 Molecular Weight
524(1)
27.4 Calculation of Molecular Weight of Oligonucleotides
524(1)
27.5 Amino Acids
525(1)
27.6 Spectrophotometric Conversions
525(1)
27.7 DNA Molar Conversions
525(1)
27.8 Protein Molar Conversions
526(1)
27.9 DNA-Protein Conversions
526(1)
27.10 Molarity
526(2)
27.10.1 Definition of Mole
526(1)
27.10.2 How to Convert Solution or Buffer Density into Moles or Molar Concentration
526(1)
27.10.3 How to Convert a Specific Volume of a Specific Concentration (100 mM) of a Solution or Buffer into Weight
527(1)
27.10.4 Common Buffer or Solution Volume Conversions
527(1)
27.10.5 Example of Preparation of a Primer Solution
527(1)
27.10.6 Example of Preparation of a Peptide Solution
527(1)
27.11 Calculation of Mean
528(1)
27.12 Variance and SD
529(1)
References
530(1)
Index 531
William Wu, Ph.D., has extensive experience in both academic and industrial settings. He held various positions, including Research Assistant Professor at the University of Michigan Medical School, Ann Arbor, Michigan, USA, and Professor at the Hunan Nonnal University, Hunan, the People's Republic of China, Senior Scientist, Team-Leader and Chief Technology Officer (CTO) in biomedical and pharmaceutical companies.

Helen H. Zhang, MS., is Senior Research Associate at the University of Michigan Medical School, Ann Arbor, Michigan, USA. In 1996, she received her MS degree in Molecular and Cellular Biology at the Eastern Michigan University, Ypsilanti, Michigan, USA.

Michael J. Welsh, Ph.D., is Professor of Cell Biology in the Department of Cell and Developmental Biology at the University of Michigan Medical School, Ann Arbor, Michigan, USA. He is also a Professor of Toxicology in the School of Public Health at the University of Michigan, Ann Arbor.

Peter B. Kanfman, Ph.D., is Professor of Biology in the Plant Cellular and Molecular Biology Program in the Department of Biology and a member of the faculty of the Bioengineering Program at the University of Michigan.