By Diana P. Bratu, Gerard P. McNeil
This quantity offers present up to date protocols for getting ready the ovary for numerous imaging thoughts, genetic protocols for producing mutant clones, mosaic research and assessing phone demise. Chapters deal with equipment for appearing genome vast gene expression research and bioinformatics for reviews of RNA-protein interactions. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and pointers on troubleshooting and averting recognized pitfalls.
Authoritative and state of the art, Drosophila Oogenesis: equipment and Protocols goals to make sure profitable leads to the extra research of this important field.
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Additional info for Drosophila Oogenesis: Methods and Protocols
Cells, which have not recombined, will be heterozygous and identical to the parental cell number of cells in intra-cyst clones. These experiments can be useful to decipher the relative contribution of the oocyte compared to nurse cells to a given process. However, these cells share the same cytoplasm through ring canals, which makes the analysis more challenging. GFP or β-gal can diffuse from one cell to another, and the genotype of each cell may not correspond to the visible marker. Secondly, mutant and wild type proteins can also pass through the ring canals, it is thus difficult to conclude on the cell autonomy or non-autonomy of a gene.
The TRIzol® Reagent can be hazardous and therefore cautious handling of this reagent is necessary in order to avoid direct contact of the skin and eyes. 3. Depending on the subsequent analysis, DNase treatment is optional during the extraction of RNA. For RNA samples that will be reverse transcribed into DNA for analysis of gene expression, DNase treatment should be done to avoid contamination of the cDNA with genomic DNA. 4. The water bath or heat block should be set at 37 °C. 5. Add a small amount of baker’s yeast to the vial of flies that will be used for dissection.
Alternative wash solutions may be used, depending on the conditions for the optimal visualization of specific proteins. 5 %) and the detergent itself (Triton™ X-100, NP-40, Tween 20, SDS) may be modified. 22. For any subsequent washes that involve fluorescent antibodies or reagents, or for ovarian samples isolated from females expressing fluorescent transgenic proteins, take care to cover the tubes with aluminum foil during all the fixation and wash steps. References 1. Mahowald A, Kambysellis M (1980) Oogenesis.