Lab book of the gilestro lab
Instructions according to Stockinger lab manual: http://www.oardc.ohio-state.edu/stockingerlab/Protocols/GlycerolStocks.pdf Make a glycerol stock of plasmid pick a single colony from the transformation plate and grow an o/n culture in appropriate medium, e.g. LB amp put 0.5 ml of 80 % sterile glycerol in sterile screw cap microcentrifuge tube add 0.5 ml of o/n culture vortex freeze @ … Continue reading [Glycerol stocks] plasmids
Immunolabelling of larval and adult brains Dissect brains in PBS Fix with 4% formaldehyde in PBS containing 0.5 EGTA, 5mM MgCl2 (see below) @RT for 25 min. Wash 3 x 5 min. (approx.) in PBST (0.3% Triton-X100 in PBS). BREAK Can store in 4% PBT @RT or 4 DEG. 4) Preincubate in 90% PBST / … Continue reading [Protocol] Immunostaining protocol from Tony (Southall)
Some helpful websites and tools for some in silico research on fly genes FlyBase, http://flybase.org/ – Ties to together resources about the individual genes, including GBrowse gene locus viewer, predicted function according to InterPro and UniProt, lists of associated papers, etc. FlyAtlas 2, http://flyatlas.gla.ac.uk/flyatlas/index.html – Gene expression profile, based on in situ hybridization (reliability?) Virtual Fly … Continue reading Fly Resources
Test 1 for different foods Standard food dried out in the course of experiment (1 week in incubator at 25 DEG, tubes prepared with food 1 1/2 week before use). Thus, tested 4 food preparations supposedly more moist than before: 1 M Glucose in 1 % Agar 3 M Glucose in 1 % Agar (Reference: … Continue reading Fly food for tubes for behavioural assays (closed)
Gel Preparation: Below shows the volume of agarose solution required to make the desired agarose gel for each unit tray size. 1. For a standard 0.7% agarose gel, add 0.7 grammes of agarose to 100 ml of 1x TAE or TBE solution. The same 1 x solution should be used in the tank buffer solution. … Continue reading Gel volume/tank @ Cleaver Scientific for reference
Tested protocols – Phenol-Chloroform extraction from F Schweisguth @ http://francois.schweisguth.free.fr/protocols/Extraction_Drosophila_total_RNA.pdf – Chloroform extraction from FlyChip @ http://www.flychip.org.uk/protocols/gene_expression/small_extraction.pdf – P Emery. RNA From Drosophila Heads, From: Methods in Molecular Biology, vol. 362: Circadian Rhythms: Methods and Protocols. E. Rosato [ed] © Humana Press Inc., Totowa, NJ Settled for the Emery Protocol for simplicity, yield and success. … Continue reading Trizol RNA Extraction Protocol: Emery Protocol w/ DNase I treatment
Protocol “Extraction of Drosophila total RNA” @ http://francois.schweisguth.free.fr/protocols/Extraction_Drosophila_total_RNA.pdf (extraction based on combination of Trizol and Phenol-Chloroform, spinning steps @ 4 DEG) Samples app. 12 male fly bodies and app. 30 heads, dissection on CO2 pad @ RT. Process Samples frozen @ – 80 DEG in PBS before extraction. Protocol as instructed, but used centrifuge at … Continue reading Phenol-Chloroform RNA extraction, Schweisguth Protocol
Protocol “Small scale extraction of total RNA from Drosophila melanogaster” @ http://www.flychip.org.uk/protocols/gene_expression/small_extraction.pdf (extraction based on combination of Trizol and Chloroform) Samples app. 30 male fly bodies. Process After incubation @ -20 DEG (step 12), sample was split in two. Sample 1 was temporarily stored @ -2 DEG in 500 ul ethanol, while sample 2 was … Continue reading Trizol RNA extraction of bodies, Flychip Protocol
Some general stuff For a reminder on how to calculate the volumes you need for the product you want to make, see, e.g., here: http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html. If your buffer is based on powdered ingredients, add the powder first and add water up to your desired volume. Put your buffer in glass bottles to autoclave later (there … Continue reading Buffer recipes