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Искусственные генетические системы. Том 1 - Патрушев Л.И.

Патрушев Л.И. Искусственные генетические системы. Том 1 — М.: Наука, 2004. — 256 c.
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and proteins .......................................... 116
3.6.4. Other reasons for low recombinant gene expression in
bacteria .............................................. 118
3.6.5. The peculiarity of the eukaryotic recombinant protein
biosynthesis in bacterial cells: inclusion bodies ..... 119
3.6.6. Purification of recombinant proteins by affinity tag
sequences.............................................. 124
3.7. Vectors for DNA transfer in animal and plant cells.......... 133
3.7.1. Expression vector pTriEx ............................. 133
3.7.2. Plant vectors......................................... 135
3.8. Recombinant DNA uptake into cells .......................... 142
3.8.1. Natural competence of bacterial cells................. 143
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3.8.2. Induction of competence in E. coli.................... 148
3.8.3. Methods and consequences of DNA uptake into cultured animal cells .......................................... 149
Chapter 4. Gene libraries ................................................. 155
4.1. Construction of gene libraries................................ 155
4.1.1. Genomic libraries...................................... 157
4.1.2. cDNA libraries ........................................ 158
4.1.3. Random, arrayed and ordered libraries.................. 160
4.2. Search of right sequences in gene libraries .................. 162
4.2.1. Use of the labeled probes.............................. 163
4.2.2. Rescreening............................................ 167
4.2.3. Subcloning ............................................ 167
Chapter 5. Recombinant gene expression systems........................... 168
5.1. Microorganisms as expression systems.......................... 168
5.1.1. Gram-negative bacteria................................. 168
5.1.2. Yeast cells as expression systems...................... 171
5.2. Cultured animal cells as expression systems .................. 174
5.2.1. Chinese hamster ovary cells (CHO line) ................ 176
5.2.2. Mouse myeloma cells (SP2/0 line) ...................... 178
5.2.3. Mouse spleen cells (MEL line).....................v.... 179
5.2.4. African green monkey kidney cells (COS lines).......... 180
5.2.5. Baculovirus-infected insect cell systems .............. 181
5.2.6. Comparing efficacy of the expression systems .......... 183
5.3. Permeabilised cells .......................................... 184
5.4. Cell-free protein synthesizing systems ....................... 185
5.4.1. Prokaryotic systems ................................... 186
5.4.2. Eukaryotic systems .................................... 189
5.4.3. Continuous-flow cell-free expression systems .......... 190
Chapter 6. Polymerase chain reaction and other methods for DNA and
signal amplification ......................................... 192
6.1. PCR in practice .............................................. 192
6.1.1. Basic principles of the PCR reaction................... 192
6.1.2. Most important components of the PCR reaction ......... 194
6.1.3. Designing the primers.................................. 195
6.1.4. Thermostable DNA-dependent DNA polymerases ............ 198
6.1.5. Other critical components and parameters of PCR
reaction................................................ 201
6.2. PCR troubleshooting........................................... 203
6.2.1. Non-specific product yields ........................... 203
6.2.2. Little or no product yield ............................ 206
6.3. PCR methods .................................................. 207
6.3.1. Standard PCR........................................... 207
6.3.2. Multiplex PCR ......................................... 207
6.3.3. Asymmetrical PCR ...................................... 207
6.3.4. Nested PCR............................................. 208
6.3.5. Hot start PCR ......................................... 208
6.3.6. Reverse-transcriptase (RT) PCR......................... 211
6.3.7. Long and accurate (LA) PCR ............................ 212
6.3.8. In situ PCR............................................ 213
6.3.9. Allele-specific PCR.................................... 214
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