Molecular Biology Techniques

Preface.Course Synopsis:Introduction.Safety Precautions.Daily Schedule.Acknowledgements.Exercises: I. DNA Preparation, Polymerase Chain Reaction, and Molecular Cloning:Cesium chloride-ethidium bromide density gradient centrifugation.PCR to synthesize virD2 flanked with restriction sites.Restriction digests of plasmid pGEX2 and PCR products.Purification of DNA fragments from agarose.Ligation of PCR product to pGEX2 vector. Transformation of E. coli with the ligated plasmid.Small-scale preparation of plasmid DNA by the alkaline lysis method.Restriction analysis.Study questions.Protein Expression, Purification, and Analysis:Expression and purification of a fusion protein.SDS-polyacrylamide gel electrophoresis.Silver stain detection of proteins.Study questions.Oligonucleotide-Directed Mutagenesis:Restriction digests of virD2 (in pCS64) and pUC119.Purification of DNA fragments from agarose.Ligatation of restriction fragment and vector.Transformation of E. coli with the ligated plasmid and recovery clones.Small-scale preparation of plasmid DNA from broth cultures.Restriction digest of DNA: examination to confirm insert.Preparation of single-stranded DNA template.Phosphorylation of oligonucleotide.Annealing mutant oligonuleotide to template. In vitro DNA synthesis by primer extension.Transform synthesis reaction into E. coli DH5a. Small-scale preparation of plasmid DNA.Confirmation of mutants by restriction analysis.Study questions.DNA Sequencing:Polyacrylamide sequencing gel electrophoresis.Didexy sequencing.Automated Sequencing.Introduction to databases and gene sequence analysis.Study questions.Southern Blot Detection of DNA:Preparation of genomic DNA from Agrobacterium tumefaciens.Restriction digestion of genomic DNA.Agarose gel electrophoresis of restriction fragments.Southern blot: denaturation and blotting of DNA gel.Preparation of probe nick translation.Hybridization and washing of Southern blots.Study questions.Northern Blot Detection of mRNA:Preparation of RNA from tobacco leaves.Agarose-formaldehyde gel electrophoresis.Northern blot: denaturation and blotting of RNA.Probe preparation. Hybridization and washing of Northern blots.Study questions.Protein Interaction Analysis in Yeast:Yeast transformation.Filter b-galactosidase assay.Index.