Abbreviations ................................................. vii
Preface ........................................................ ix
Chapter 1. Understanding recombinant DNA techniques ............. 1
1.1. The in vitro workbench .......................... 1
1.2. When the goal is a clone ........................ 4
Chapter 2. The recombinant DNA laboratory ...................... 11
2.1. Supplies ....................................... 11
2.2. Reagents ....................................... 17
2.3. Technique ...................................... 24
2.4. Looking at DNA on a gel ........................ 35
2.5. Safety ......................................... 49
2.6. Documentation .................................. 58
2.7. Special lab problems ........................... 61
Solutions to problems ............................... 66
Chapter 3. Solutions, buffers, stocks and cocktails ............ 67
3.1. Solutions, suspensions and slurries ............ 67
3.2. Solubility of macromolecules ................... 69
3.3. Solute concentration ........................... 72
3.4. Solution pH .................................... 75
3.5. Precipitation methods .......................... 76
3.6. Preparing a solution ........................... 81
3.7. Buffer considerations .......................... 88
3.8. Stock solutions ............................... 100
Solutions to problems .............................. 105
Chapter 4. Cloning vectors .................................... 109
4.1. Plasmids ...................................... 109
4.2. Viruses ....................................... 147
4.3. Chimeras and artificial chromosomes ........... 161
4.4. Common applications ........................... 167
Solutions to problems .............................. 181
Chapter 5. Restriction endonucleases .......................... 185
5.1. Recognition sequences ......................... 185
5.2. Cleavage sites ................................ 189
5.3. Reaction conditions ........................... 198
5.4. Restriction mapping ........................... 206
Solutions to problems .............................. 216
Chapter 6. Polymerases ........................................ 221
6.1. Overview ...................................... 221
6.2. DNA labeling .................................. 230
6.3. Site-specific mutagenesis ..................... 234
6.4. DNA sequencing ................................ 242
6.5. Reverse transcriptases ........................ 257
6.6. Terminal deoxynucleotidyl transferases ........ 268
6.7. RNA polymerases ............................... 270
6.8. The special problem of specific activity ...... 275
Solutions to problems .............................. 283
Chapter 7. The polymerase chain reaction ...................... 291
7.1. Overview ...................................... 291
7.2. Primer design ................................. 292
7.3. Cycle efficiency .............................. 306
7.4. Setting up a reaction ......................... 316
7.5. Mutagenesis and engineering ................... 325
7.6. Diagnostic methods ............................ 353
7.7. Real time or quantitative PCR ................. 356
7.8. Troubleshooting ............................... 360
Solutions to problems .............................. 367
Chapter 8. Working with DNA: the reasons ...................... 375
8.1. Engineering DNA sequences ..................... 375
8.2. Identifying DNA sequences ..................... 381
8.3. Functioning of DNA sequences .................. 393
Index ......................................................... 401
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