实验动物技术

FREEZING WORM STOCKS1. Grow 3-5 6cm. plates of worms on NGM with OP50 until the plate is starved with lots of young larvae. The L1s are the ones that survive freezing the best and are the most likely ...

Freezing Worms by Michael Koelle 4/6/94 I. The preferred method 1. Wash worms off of 1 large plate or 3 small plates in 3 mls S Basal into a sterile 15 ml disposable centrifuge tube. Worms should be h ...

Growing worms in liquidFirst make worm food -- grow liters of bacteria Inoculate each of several liters (4 is a good number) of LB in 2 liter flasks with 1 ml of an overnight culture of AMA1004. Grow ...

Lineaging of C. elegans Embryosby Pressure-free MountingMaterials:Glass Slides 18mm x 18mm Coverslips Petroleum Jelly #15 Disposable Scalpel Embryonic Blastomere Medium ...

Integrating extrachromasomal arrays into the C. elegans chromosomes Why and how to do it by Michael Koelle 4/6/94 What is the benefit of integrating an extrachromosomal array? Extrachromosomal arrays ...

Lethal phase determination1. Pick a bunch of heterozygote L4 hermaphrodites to a new plate.2. The next day there will be eggs on the plate; pick exactly 20 of these to each of a few plates. Make sure ...

Integrating extrachromasomal arrays into the C. elegans chromosomesWhy and how to do itby Michael Koelle12/20/94What is the benefit of integrating an extrachromosomal array? Extrachromosomal arrays su ...

Microinjecting worms by Michael Koelle8/23/94You will have a couple frustrating sessions when you first attempt this technique but everyone seems to master injection after a few days and it works very ...

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 ...