Trailblazers: Investigating Chemotaxis with C.elegans
Module III: Collection of C.elegans and Chemotaxis Assay Excerpts from Edvo-Kit #852
1. LABEL one 15 mL tube of S-Buffer, a large transfer pipet, and a snap top tube with “WT” (Wild- Type) and the other 15 mL tube, large transfer pipet, and snap top tube with “MS” (Mutant Strain). 2. Using the WT transfer pipet, TRANSFER 3.5 mL of S-Buffer from the WT conical tube to the Petri dish containing the wild-type C. elegans . 3. DISLODGE worms by rinsing the dish several times. Rinse the dish by either (a) swirling the plate or (b) holding the plate at a slight angle and allowing the buffer to collect near the bot - tom. Next suck up the buffer using the transfer pipet and then expel the buffer near the top so that it runs down the plate. 4. Once most worms are suspended in the buffer, TRANSFER the worms and buffer back to the WT 15 mL conical tube using the WT transfer pipet. 5. REPEAT steps 2-4 for mutant C. elegans using the MS labeled items. 6. Keep the conical tubes still and upright to ALLOW the worms to settle to the bottom of the tubes (~10 minutes). While you wait, CONTINUE to steps 7, 8, and 9. 7. PREPARE two NGM plates for the assay using the template provided (see Figure 6, page 15). a. TRACE the two inner lines and then use a well cutter to punch the three holes. b. REMOVE the NGM plugs from each hole with a flat edged toothpick or spatula. c. LABEL each plate with either “WT” or “MS” on the side and on the lid. 8. To both assay plates, ADD 30 µL of your experimental compound to the left well and 30 µL of the control solution to the right well. RECORD which chemical compound was used here and in your lab book.
Experimental Compound: _________________________.
9. (OPTIONAL) ADD 10 µL of Sodium Azide to the two outer wells of both plates. BE CAREFUL AND WEAR GLOVES WHEN HANDLING BOTH THE SODIUM AZIDE TUBE AND THE PLATES WITH SODIUM AZIDE . 10. Using the appropriately labeled transfer pipets, slowly REMOVE ~3 mL of the cleared S-Buffer supernatant without disturbing the worms that have settled to the bottom of the tubes. Be- tween 300 µL and 500 µL of buffer with worms should remain at the bottom of both tubes. 11. TAP both tubes several times to resuspend the worms.
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