实验动物模型

Generating males by heat shockby Michael Koelle1. Set up ~6 plates with 5 L4 hermaphordites each.2. Heat shock 4-6 hours at 30°. 8-9 hours works but gives few progeny.3. Return to 20°. Should get a fe ...

Making Dauers with dauer pheromoneAuthor: Sho Gottlieb 1992 1) Make tiny plates with NGM-peptone worm plate recipe 2) 2%(2g/100ml) DHSalpha cells O/N culture(are not strep resistant e.coli) 3) Add str ...

Liquid culture of wormsBy Michael Koelle and Tory Herman adapted from Mir Hengartner4/6/94Media1) superbroth (5 X 2 1/2 liters autoclaved in 6 liter flasks) 5 X 30 g Bactotryptone 5 X 60 g yeast extra ...

N2 development times at different temperaturesby Michael KoelleDetermined empirically; times in hours are given from the first division of the zygote.15°20°25°eggs laid217hatch22139L1 molt492921L2 mo ...

Worm genomic Southern blotsby Michael Koelle4/6/94I. Preparing worm genomic DNA: requires 1-2 days to seed agarose plates a few days for the worms to grow 1-2 days to prep the DNA1. Seed large agarose ...

Worm PCRPick one worm and place it in a 2.5 l drop of lysis buffer in the cap of a PCR tube. Close and centrifuge briefly to move to the bottom of the tube. Freeze the tubes at -70°C for 15 min. The ...

Genomic DNA prep(Jorgensen Lab ) Make worm growth plates. These use agarose to avoid impurities in most batches of agar and are enriched to allow greater worm growth. Mix:5 g Bacto tryptone2 g Bacto y ...

Joe's mRNA prep(Audrey Gasch Pat Brown ) Oligo-dT cellulose prepDump Ambion vial contents into 50 ml c/f tube Add 10 ml 1x NETS to vial cap rinse vial dump 10 ml into c/f tube Spin tube 3000 rpm 2 min ...

Yale RNA prep(Rebecca D. Burdine Michael J. Stern ) Add 8 ml of TRIZOL to 2 ml packed worms in 15 ml centrifuge tube. Vortex and invert tube to solubilize and lyse worms for at least 10 min. Divide in ...

Immunofluorescence of C. elegans EmbryosMaterials:fixative dependent on antigen (common: 4% formaldehyde or 100% MeOH) post-fixative dependent on antigen (usually MeOH) PBS 1X PBS PBST PBS + detergent ...

DAPI Staining To visualize DNA incubate fixed samples with 100 ng/ml 4'6'-diamidino-2-phenylindole hydrochloride (DAPI) in PBS for 30 min. Rinse 3x with PBTw. MaterialsPBS: Sambrook et al. (Molecular ...

Double dye filling (using DiO and di-4 ANEPPS)(Krista Williams) Dye Filling Label 15 ml centrifuge (c/f) tubes with strain name. Squirt ~1-2 ml M9 onto plate with a Pasteur pipet. Rinse plate with M9 ...

Dye Filling to Stain Amphid and Phasmid Neurons by Michael Koelle from Beth Sawin 4/6/94 1. Buy DiO from Molecular Probes catalog # D-275. DiO fluorescesces green (use FITC filters). If you want red f ...

Fixation and Staining ParametersAntigen Fixation Fixation time AntibodyDilutionRefmicrotubules 100% MeOH 5 min @ RT DM1a1:500actin 4% Formaldehydundefined 8 min @ RT C41:5001POD-1 Formaldehyde or MeOundefined 8 min ...

EM Fixation of EmbryosAdapted following advice from Andy Fire and Jim Priess1. Mount embryos on slides as if for DIC except agar has fixative. Agarose Fixative 3ml 2% Agarose : 1.5% LGT Agarose + .05% ...

Bloomington No-Plug Food Prep Method Standard medium in use at Bloomington Hard agar medium in use at Bloomington Cornmeal Sucrose Dextrose Yeast and 2-Acid Medium Cornmeal-molasses-yeast medium Co ...

Gonad Dissections- From R. Francis -1) Pick adults and/or L4s to an unseeded NGM plate. Alternatively wash worms off a seeded plate with 1.5 ml of PBS and allow to gravity settle for a few minutes in ...

Gonad Fixation - From R. Francis -A. Methanol Methanol fixation is quick easy and gives satisfactory nuclear morphology after DAPI staining. However methanol-fixed gonads break easily and are more dif ...

In situ hybridizationIn situ hybridizations were based on a modified method of Seydoux and Fire (1994 Development 120 2823-2834; 1995 Methods in Cell Biology) (H. Tabara and Y. Kohara personal commun ...

FREEZE-CRACK b-GALACTOSIDASE STAINING PROTOCOLFIRE LAB1. Prepare humidity chamber: line large square plate with humid paper towels and place 4 5ml plastic pipets across chamber to support slides.Place ...