BISC219/F12: RNAi Lab 10
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Lab 10: Series 3 Investigation of Gene Regulation Using RNAi--
Preparation of the Na+ gradient in the Chemotaxis Assay media
The day before lab your instructor will add the chemicals needed to prepare the chemotaxis assay plates.
- 10 ul of 2.5M NaCl is added to the Na dot you drew on your plate. Once the NaCl is absorbed the Na+ ions will disperse in a gradient away from the dot. The Cl2 is not in a gradient due to the addition of MgCl2 to the media during initial preparation.
- 10 ul of sterile water is added to the W dot you drew to serve as a negative control.
- Additional control plates will have 10 ul of sterile water added to both dots on the plate.
Harvesting the C. elegans
- Label 3 15 ml conical tubes with wild type - treated
- Label 3 15 ml conical tubes with rrf-3- treated
- Label 3 15 ml conical tubes with wild type - control
- Label 3 15 ml conical tubes with rrf-3 - control
- Wash the worms off of each RNAi feeding plate 3x with 5 ml sterile water - putting wash in a new labeled 15 ml tube each time - store the tubes with worms in them on ice.
- Once all the worms are collected, put the caps on the tubes and invert a few times to mix the worms.
- Let the worms settle to the bottom of the tubes - about 10 minutes
- Remove all but 1 ml of the water with a disposable Pasteur pipette.
- Add sterile water to 15 ml and cap the tube again. Invert and let the worms settle.
- Remove all but 1 ml of the water with a disposable Pasteur pipette.
- Transfer the remaining 1 ml of water + worms to a sterile 1.5 ml microfuge tube.
- Spin the worms at 10,000 rpm for 1 minute to pellet.
- Remove all but approximately 50 ul of worms and water at the bottom of the tube.
- Using a razor blade, cut the end off of a micropipette tip (this will prevent the pellet of worms from being damaged).
- Pipette the entire pellet of worms to the appropriate assay plate.