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Tables of Contents for Plant Molecular Biology
Chapter/Section Title
Page #
Page Count
Abbreviations
x
3
Preface
xiii
1
Safety
xiv
I Isolation of proteins and nucleic acids
1
43
Isolation of protoplasts
1
1
Isolation of plant organelles: chloroplasts, nuclei, mitochondria and peroxisomes
1
1
Isolation of nucleic acids
1
1
Isolation of specific RNA types or species using affinity methods
2
1
Isolation of genomic DNA
3
1
Isolation of proteins
4
1
Construction of DNA libraries
5
2
Methods available
7
Protoplast isolation
7
1
Isolation of plant organelles
7
1
Isolation of mRNA with oligo(dT)-cellulose
8
1
Genomic DNA isolation
8
1
Chloroplast DNA isolation
8
1
Protein isolation
9
1
cDNA library construction
9
1
Genomic DNA library construction
10
References
7
5
Protocols
12
32
1 Isolation of protoplasts - optimized for young barley
12
4
2 Isolation of plant organelles
16
4
3 Messenger RNA isolation
20
4
4a Genomic DNA extraction
24
2
4b Chloroplast DNA isolation
26
2
5 Isolation of proteins from plant tissue
28
2
6 Construction of a cDNA library
30
6
7a Preparation of partially digested DNA for construction of a genomic DNA library
36
4
7b Ligation of size fractionated genomic DNA into lambda vector arms
40
2
7c Packaging and titration of a genomic DNA library
42
2
II Analysis of gene expression: gene structure
44
18
Radiolabeling DNA
44
1
Southern blot analysis
44
1
Random amplified polymorphic DNA (RAPD) analysis of genomic DNA
44
1
Gel retardation
45
2
Basic principles
45
1
Binding conditions
46
1
Electrophoresis and analysis
46
1
Competitor analysis of nucleic acid-protein interactions
46
1
DNA-protein and RNA-protein interactions
47
1
S1 mapping
47
1
Methods available
47
1
PCR production of [(32)P]-CTP-labeled fragments
47
1
Southern blot analysis
47
1
RAPD analysis of plant genomic DNA
48
1
Gel retardation of rRNA-RNA processing complex
48
1
S1 mapping
49
References
47
3
Protocols
50
12
8 PCR production of [(32)P]-CTP-labeled fragments
50
2
9a Capillary blotting on to nitrocellulose or nylon membranes
52
2
9b Southern probing for low and high abundance targets
54
2
10 Random amplified polymorphic DNA (RAPD) analysis of plant genomic DNA
56
2
11 Gel retardation of rRNA-RNA processing complex
58
2
12 Nuclease S1 analysis
60
2
III Analysis of gene expression: transcription
62
56
Introduction
62
1
Northern analysis
62
3
Reverse transcriptase-polymerase chain reaction: RT-PCR
65
1
Differential display
65
1
High stringency cDNA substraction
66
1
Nuclear run-ons
67
1
Primer extension analysis
67
1
RNA protection assays/RNA mapping
67
1
In situ hybridization
67
2
Methods available
69
3' End labeling of RNA
69
1
Northern analysis
69
1
Reverse transcriptase: polymerase chain reaction (RT-PCR)
70
1
Differential display
70
1
Nuclear run on analysis
71
1
High stringency subtractive PCR
71
1
Primer extension analysis
72
1
Riboprobe preparation by in vitro transcription
73
1
In situ hybridization
73
References
69
Protocols
74
44
13 3' End labeling of RNA using T4 RNA ligase
74
2
14a Northern analysis: agarose gel electrophoresis of RNA
76
2
14b Northern analysis: probing membrane bound RNA
78
2
15 Reverse transcriptase-polymerase chain reaction (RT-PCR)
80
4
16 Differential display
84
4
17 High stringency subtractive polymerase chain reaction (PCR)
88
6
18 Nuclear run-on analysis
94
4
19 Primer extension analysis
98
4
20a RNase protection assay: riboprobe preparation by in vitro transcription
102
2
20b RNase protection assay: hybridization, digestion and analysis
104
2
21a Tissue preparation for in situ hybridization
106
4
21b Preparing a digoxygenin labeled riboprobe for in situ hybridization
110
2
21c In situ hybridization
112
4
21d Development and mounting of in situ hybridized tissue for examination
116
2
IV Analysis of gene expression: protein analysis
118
18
In vitro translation
118
1
Use of antibodies for detection of proteins
119
1
Immunological detection of membrane-bound antigens
119
2
Immunolocalization
121
2
Methods available
123
In vitro translation
123
1
Staining of immunoblots
123
1
Immunolocalization
124
References
123
3
Protocols
126
10
22 In vitro translation -- wheat germ system
126
2
23a Detection of antigens on western blots using alkaline phosphatase-conjugated antibodies
128
2
23b Detection of antigens using horseradish peroxidase-conjugated antibodies and enhanced chemiluminescence (ECL)
130
2
24a Fixation and sectioning of plant tissue for immunolocalization
132
2
24b Gold labeling of tissue sections, with silver enhancement
134
2
V DNA manipulation
136
30
Site-directed mutagenesis
138
1
Promoters
138
2
Transcriptional and translational gene fusions
140
2
Protein trafficking
142
1
Expression levels
143
1
Vectors
144
1
Selectable markers and reporter genes
144
1
Methods available
145
DNA cloning
145
1
Site-directed mutagenesis
146
2
Calcium chloride-mediated transformation
148
1
Electroporation
148
1
Triparental mating
149
6
References
145
5
Protocols
150
16
25 Cloning of DNA fragments
150
4
26 Site-directed mutagenesis
154
4
27a Calcium chloride-mediated transformation of Escherichia coli
158
2
27b Calcium chloride-mediated transformation of Agrobacterium
160
2
27c Transformation of Agrobacterium tumefaciens by high-voltage electroporation
162
2
28 Triparental mating of binary vectors into Agrobacterium
164
2
VI Transformation, transfection and regeneration
166
40
Introduction
166
1
Agrobacterium-mediated plant transformation
166
2
Direct gene transfer
168
3
Protoplast transformation and transfection
168
2
Direct gene transfer into intact tissue
170
1
Analysis of transformation
171
2
Insertion of T-DNA
171
1
Gene expression
171
2
Methods available
173
Leaf disk transformation using Agrobacterium tumefaciens
173
1
Root transformation
173
1
Agrobacterium rhizogenes transformation of plants
173
1
Protoplast transfection
174
1
Protoplast transformation and regeneration
174
1
Particle bombardment
175
1
PCR analysis of transformation
175
1
Reporter gene assays
176
References
173
5
Protocols
178
28
29 Transformation of potato leaf disks using Agrobacteriun tumefaciens
178
4
30 Root transformation of Arabidopsis thaliana
182
4
31 Plant transformation using Agrobacterium rhizogenes
186
2
32 Protoplast transfection by electroporation
188
2
33 Polyethylene glycol-mediated protoplast transformation (potato)
190
4
34 Biolistic transformation of maize suspension cultures
194
4
35 Polymerase chain reaction (PCR) analysis of plant transformation
198
2
36a BETA-D-Glucuronidase (GUS) fluorescence assay
200
36b BETA-D-Glucuronidase (GUS) chemiluminescence assay
202
2
36c Luciferase assay
204
2
Appendix: Suppliers
206
7
Index
213