Molecular Biology Techniques: An Intensive Laboratory Course
Regular price
32.250 KD
inc. VAT
Couldn't load pickup availability
Table of contents
- Cover
- Copyright Pageiv
- Contentsv
- Prefaceix
- Course Synopsis1
- Introduction2
- Safety Precautions7
- Daily Schedule9
- Acknowledgments15
- Exercises I. DNA Preparation, Polymerase Chain Reaction, and Molecular Cloning17
- A. Cesium chloride–ethidium bromide density gradient centrifugation19
- B. PCR to synthesize virD2 flanked with restriction sites27
- C. Restriction digests of plasmid pGEX2 and PCR products35
- D. Purification of DNA fragments from agarose39
- E. Ligation of PCR product to pGEX2 vector45
- F. Transformation of E. coli with the ligated plasmid49
- G. Small-scale preparation of plasmid DNA by the alkaline lysis method55
- H. Restriction analysis59
- Study questions63
- Exercises II. Protein Expression, Purification, and Analysis71
- A. Expression and purification of a fusion protein75
- B. SDS-polyacrylamide gel electrophoresis81
- C. Silver stain detection of proteins87
- D. Western blot (immunoblot) detection of proteins91
- Study questions97
- Exercises III. Oligonucleotide-Directed Mutagenesis99
- A. Restriction digests of virD2 (in pCS64) and pUC119109
- B. Purification of DNA fragments from agarose111
- C. Ligation of restriction fragment and vector113
- D. Transformation of E. coli with the ligated plasmid and recovery of clones117
- E. Small-scale preparation of plasmid DNA from broth cultures117
- F. Restriction digest of DNAs: Examination to confirm insert117
- G. Preparation of single-stranded DNA template119
- H. Phosphorylation of oligonucleotide125
- I. Annealing mutant oligonucleotide to template129
- J. In vitro DNA synthesis by primer extension133
- K. Transform synthesis reaction into E. coli DH5α137
- L. Small-scale preparation of plasmid DNA137
- M. Confirmation of mutants by restriction analysis138
- Study questions141
- Exercises IV. DNA Sequencing143
- A. Polyacrylamide sequencing gel electrophoresis147
- B. Dideoxy sequencing151
- C. Automated sequencing155
- D. Introduction to databases and gene sequence analysis157
- Study questions161
- Exercises V. Southern Blot Detection of DNA163
- A. Preparation of genomic DNA from Agrobacterium tumefaciens165
- B. Restriction digestion of genomic DNA169
- C. Agarose gel electrophoresis of restriction fragments171
- D. Southern blot: Denaturation and blotting of DNA175
- E. Preparation of probe by nick translation179
- F. Hybridization and washing of Southern blots185
- Study questions189
- Exercises VI. Northern Blot Detection of mRNA191
- A. Preparation of RNA from tobacco leaves195
- B. Agarose–formaldehyde gel electrophoresis199
- C. Northern blot: Denaturation and blotting of RNA203
- D. Probe preparation207
- E. Hybridization and washing of Northern blots211
- Study questions215
- Exercises VII. Protein Interaction Analysis in Yeast217
- A. Yeast transformation221
- B. Filter β-galactosidase assay225
- Index229
This manual is designed as an intensive introduction to the various tools of molecular biology. It introduces all the basic methods of molecular biology including cloning, PCR, Southern (DNA) blotting, Northern (RNA) blotting, Western blotting, DNA sequencing, oligo-directed mutagenesis, and protein expression.
Key Features
* Provides well-tested experimental protocols for each technique
* Lists the reagents and preparation of each experiment separately
* Contains a complete schedule of experiments and the preparation required
* Includes study questions at the end of each chapter
Key Features
* Provides well-tested experimental protocols for each technique
* Lists the reagents and preparation of each experiment separately
* Contains a complete schedule of experiments and the preparation required
* Includes study questions at the end of each chapter