C. Elegans Unit: Lab Activities

Overview of Techniques Basic Worm Handling Techniques www.silencinggenomes.org Pouring NGM Agar plates Nematode Growth medium Innoculating broth with OP50 E. coli for feeding “Seeding” NGM Agar plates with OP50 Chunking worms to propagate more worms Picking worms to move exact numbers of worms Making a worm pick Chemotaxis Assay – Biorad Kit

Basic Technique: Pouring NGM Plates Melt down 1-2 bottles of NGM agar Hot plate, water bath, microwave About 14 plates per group of 2 Label with Initials Date NGM agar INSTRUCTOR NOTE: HAVE a student pour 16 extra plates for instructor use with Chemotaxis kit (worm thawing) Instructor Prep: Petri Dishes, NGM, sharpies

Basic Technique: Innoculating with OP50 Using sterile technique, transfer 3mls of LB broth (premade) to a clean bacterial culture tube (the clear ones with loose caps) Dip a sterile innoculating loop into the OP50 stock broth or touch to surface of stock plate culture Put loop into your LB broth and swirl Label with your initials, date, and OP50 Put in tube racks in 37°incubator NOTE: All materials that touch bacteria go in bacterial waste pans Instructor prep: Thaw OP50 stock, get LB broth (premade – if not left make some, autoclave it), Set up bleach pans

Basics: Seeding NGM plates with OP50 Add OP50 in red sharpie to the labels on your plates Once OP50 has grown for at least 24 hours, Add 200 μl of OP50 broth culture to the surface of 6 of your NGM plates (2 for chunking, 2 for picking, 2 for making worm stocks for chemotaxis assay later) Leave any remaining plates empty for later –store in fridge Using an L-spreader, spread the broth around, avoiding the very edges of the dish. Let soak in about 15 min, then parafilm Invert and incubate at ROOM TEMP. NOTE: All materials that touch bacteria go in bacterial waste pans Instructor prep – Bacterial waste pans

Basic Technique: Chunking worms Obtain a stock plate of N2 (wild-type) worms from instructor Get 2 of your NGM + OP50 plates, Add to the labels “N2” Flame-sterilize a scalpel, and touch to surface of agar to cool Cut a 1cm2 square of agar from the stock plate Transfer it to the surface of the NGM-OP50 plate Parafilm and invert Incubate at room temperature Instructor prep: scalpels, lighters

Basic Technique: Picking Worms Instructor will demonstrate how to make a worm pick Make your worm pick and label it with your initials Get a plate of worms that was chunked a few days ago and has many worms on it Transfer 5-10 worms to an NGM + OP50 plate Practice! Video: https://www.youtube.com/watch?v=eTQtkQm5 hOw&feature=related Instructor prep: Platinum wire, clay, pliers

Biorad Chemotaxis Assay: Quick Guide Pour NGM agar plates, Seed with OP50 To make stocks of N2 and Mutant worms for assay Use Wash buffer (S-buffer) method to transfer worms to make a new stock plate Pour assay plates - 20 total per kit Prepare Assay plates Perform assay Collect & record data Perform Statistical analysis

Biorad Kit Prepare NGM seeded with OP50 You have extra from our basic worm techniques – you’ll need just 2 plates for this assay You should have 2 plates left with NGM & OP50, no worms – if not seed more. NOTE: the kit calls for use of OP50-pBAD which has an amp resistance gene, and requires culture on NGM+amp. We are skipping this as it is not necessary and gives us more flexibility. Instructor Prep: None

Biorad Kit: Pour assay plates These have a small amount of Wash buffer in the agar instead of NaCl (a component of NGM) We have just 20 per class (per kit) Instructor has prepped solution & autoclaved it; Each team come pour 2 plates Label with your initials, date and ASSAY plates Instructor prep: Make the assay buffer from kit – comes powdered – see instruction in kit, Autoclave it.

Biorad Kit: Making stocks of WT and Mut (daf-18) worms Use wash buffer transfer method (S-buffer technique) to make new stocks of your worms onto NGM + OP50 (NOT ASSAY PLATES) Follow instructions on pp. 30-31 of your kit manual Make sure you label plates with WT worms (N2) or Mutant (daf-18) Don’t cross contaminate them Be sure to incubate at room temperature Need 5 days to make enough worms for next steps Instructor prep: Call in Biorad kit coupon in advance to to get 2 worm strains, then when they arrive, thaw and plate onto fresh NGM (see kit) – no OP50 is necessary on plates, frozen worm stocks have it. The worms need to grow for 4-7 days before they can be used by students. Dilute wash buffer.

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BioRad Kit: Finishing assay plates – adding salt gradient Instructor has prepared wash buffer and 2.5M NaCl solution Follow instructions on next slide to set up plates Let plates incubate overnight before use. Instructor Prep: Dilute wash buffer

BioRad Kit: Chemotaxis Assay Follow the instructions on pp. 35 -38 of your handout to set up chemotaxis assay Allow 30 minutes for worms to move Collect data & record in notebook Next class: Perform Statistical analysis

Chi Square Analysis Follow along with BioRad kit Chi-Square supplement Read it Try practice problems Analyze your own data Record in notebook