生物化学技术

DROSOPHILA HEAD COLLECTION(Baker Lab) Equipment flies frozen in 250 ml centrifuge bottlessieves: 106, 355, 600 and 850 mmpaintbrushcollection tubesfunneldry icejacket, hat and gloves! Preparation 1. Freeze flies in 250 ml centrifuge bottle(s) @ -80oC („30').2. Cool sieves @ -20oC for „ 20' p ...

DROSOPHILA DNA PREP(essentially Roberts) 1. Grind 20-50 frozen flies with a mortar and pestle in N2(l) and suspend in 2 ml Lysis Buffer. 2.Add Proteinase K to 100 mg/ml; incubate 1-2h @ 37oC with occassional swirling. 3. Phenol extract GENTLY, 10'; cfg. 5' @ RT. Harvest the aqueous phase with a large bore transfer p ...

Preabsorbing Antisera with C. elegans Acetone Powderby Michael Koelle, adapted from "Antibodies: A Practical Approach" by D. CattyOne way to eliminate crossreactivity of a polyclonal antiserum raised against a worm protein is to preabsorb the antiserum against total protein extra ...

Dye Filling to Stain Amphid and Phasmid Neuronsby Michael Koelle, from Beth Sawin1. Buy DiO from Molecular Probes, catalog # D-275. DiO fluorescesces green (use FITC filters). If you want red fluorescence, DiI can be substituted; stain exactly the same way but use the Texas red filters on the fluoresc ...

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 to use first day eggs like this: wild-type animals eventually run out of sperm and lay unfertilized eggs that don't d ...

Integrating extrachromasomal arrays into the C. elegans chromosomesWhy and how to do itby Michael KoelleWhat is the benefit of integrating an extrachromosomal array? Extrachromosomal arrays suffer from three problems that may be solved to different extents by integration into the ...

Liquid culture of wormsBy Michael Koelle and Tory Herman, adapted from Mir HengartnerMedia1) superbroth (5 X 2 1/2 liters autoclaved in 6 liter flasks) 5 X 30 g Bactotryptone 5 X 60 g yeast extract 5 X 20 mL 50% glycerol stock solution 5 X 2.25 liters DDWHint: use a funnel to pour measured ingredients into the flasks. ...

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 quickly and reliably once you have some experience.1. Materialsa) agarose pads: make a lot ...

Response to food assayby Meng-Qiu Dong 1/1/2000Wild type worms adjust egg-laying behavior in response to food. They lay eggs when they are fed and stop laying eggs when they are starved. If starved wild type worms are put back on food, they resume egg laying almost immediately. I use the following assay to de ...

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 molt64.53827L3 molt804733L4 molt98.55841For staging later anim ...

Freezing Wormsby Michael KoelleI. The preferred method1. 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 harvested off of just starved plates which are predominantly L1 and L2. This should be 1 day after the bacteria have been exh ...

Culturing Wormsby Michael Koelle1. Bacterial strain for feeding worms: OP50, a uracil auxotroph. Streak OP50 out on a 9 cm NGM agar plate , grow overnight at 37°. Can then parafilm the plate and keep it at 4° for months. To make bacteria for seeding plates, use a flamed wire loop to pick a single OP50 colony into a 100 ml bo ...

Cleaning Worm Stocksby Michael KoelleThere are two kinds of contaminants on worm plates:1. Fungi: these contaminants can come from the plates or bacteria, so it is best to leave plates out after seeding for a couple of days to make sure no fungal colonies grow before adding worms. If a stock gets mold, just tr ...

秀丽线虫RNA提取 C. elegans RNA prepby Michael Koelle and Tory Herman, adapted from Sambrook et al., "Molecular Cloning"C. elegans RNA preps that have been widely used previously involved fairly slow lysis of the worms, potentially leading to degradation of the RNA. In addition, they failed to separa ...

线虫基因组杂交 Worm genomic Southern blotsby Michael KoelleI. 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 plates with HB101. Agarose is preferred over agar since the latter contains more inhibitors of rest ...

Making Protein Gel Samples from Wormsby Michael Koelle1. Grow up 3-5 large plates of worms. This should give ~200 µl of packed worms after the washes described below.2. When the worms have almost or have just starved, wash the worms off the plate by dumping a little M9 medium on the plate, swirling gently, and su ...

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 few males per plate in the F1.4. When you only have a few males , it is best to set them up with an excess of L4 hermaphrodites to ensure ...

Preparing Serum from Whole Bloodby Michael Koelle, from Harlow and Lane "Antibodies: A Laboratory Manual"1. Veterinary services people often will do bleeds for you and give you the fresh blood in glass vacutainer tubes, leaving you to prepare serum from it.2. Put the blood at 37° for 30-60 min to clot.3. P ...

Please click on the link below to download the protocol.This protocol can be downloaded as a Microsoft WORD document.However, it is password protected and it cannot be modified.C. elegans Gene Knockout Protocol 上一篇：Preparing Serum from Whole Blood(worms) 下一篇：Simplified Arabidopsis ...

Arabidopsis RNA extraction protocolBased on Krapp at al (1993) Plant Journal 3:817 with modifications.Comments or Questions? Send to me at: chunming.liu@wur.nl1-2 g fresh material, freezer-dried, ground with 0.2g sand (if necessary), and then homogenized with 10ml RNA extraction buf ...