By Florence Malka, Karine Auré, Steffi Goffart, Johannes N. Spelbrink, Manuel Rojo (auth.), Dario Leister, Johannes M. Herrmann (eds.)
Mitochondria: functional Protocols bargains a wide choice of tools for learning the molecular biology, functionality, and lines of mitochondria. some time past decade, mitochondrial examine has elucidated the $64000 impression of mitochondrial strategies on indispensable phone approaches similar to apoptosis and mobile getting older. This useful consultant offers a large spectrum of mitochondrial tools, each one written by means of experts with reliable event and meant for implementation via amateur and specialist researchers alike.
Part I introduces significant experimental version platforms and discusses their particular benefits and barriers for sensible research of mitochondria. The concise evaluation of basic houses of mitochondrial platforms is supplemented by way of distinctive protocols for cultivation of version organisms. elements II-VI contain a strong selection of protocols for learning assorted molecular facets of mitochondrial capabilities together with: genetics and microbiology, biochemistry, body structure, dynamics and morphology, and useful genomics. Emphasis is put on new and rising themes in mitochondrial research, akin to the exam of apoptotic results, fusion and fission of mitochondria, and proteome and transcriptome analysis.
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Extra resources for Mitochondria: Practical Protocols
5. , 300 rpm in a shaker). 6. Remove the formaldehyde phase (the lower phase). 7. Add 10 mL methanol and mix vigorously by hand for 60 s. This removes the embryonic vitelin membrane. 8. Embryos sediment in a few seconds. Those with vitelin membranes remain suspended. 9. Remove everything except embryos at the bottom. 10. Add 5 mL methanol, mix gently, and remove it. Repeat twice. 11. 5-mL tube, and store at 4°C or 20°C (see Note 19). 3. Hybridization, Developing, and Visualization Although embryos are already fixed, we recommend fixing the embryos again after storage.
2. Mitochondrial Extraction 1. 20% (w/v) Sodium cholate. Cholic acid is dissolved in hot ethanol, filtered through Norit A (J. T. 4 with sodium hydroxide. 03_Fernández_Moreno 4/2/07 11:29 AM Page 42 42 Fernández-Moreno et al. 2. 3 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 mM sodium metabisulfite, 2 Rg/mL leupeptin. 3. 0, 2 mM EDTA, 80% (v/v) glycerol. 4. 5 M Sodium chloride. 5. 5-mL Microcentrifuge tubes. 6. 1. 3. 1. Preparation of the Probe 1. For transcription, we use the in vitro labeling kit from Roche (DIG RNA Labelling Kit SP6/T7; cat.
4. HOMOLOGOUS RECOMBINATION Although historically it was thought that Drosophila lacks the homologous recombination process, the method developed by Golic and collaborators demonstrated that it does occur (7). This technique allows precise substitution of a specific DNA region of the Drosophila genome by another homologous, although not identical, sequence. 2. Overexpression Phenotype ( see Chapter 15) By a relatively easy transgenesis, one can introduce extra copies of a complementary DNA or a gene under the control of a selected promoter.