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.

  1. 10 μL 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.
  2. 10 μL of sterile water is added to the W dot you drew to serve as a negative control.
  3. Additional control plates will have 10 μL of sterile water added to both dots on the plate.

Harvesting the C. elegans
Thoroughly washing the worms to remove any residual food and media is critical for the chemotaxis assay to work properly.

  1. Label 2 15 ml conical tubes with wild type - treated
  2. Label 2 15 ml conical tubes with rrf-3- treated
  3. Label 1 15 ml conical tubes with wild type - control
  4. Label 1 15 ml conical tubes with rrf-3 - control
  5. Label 2 15 ml conical tubes with lsy-2 mutant
  6. Wash the worms off of each RNAi feeding plate 3x with 5 ml sterile water - putting each wash in the correct labeled 15 ml tube - store the tubes with worms in them on ice.
  7. Once all the worms are collected, put the caps on the tubes and invert a few times to mix the worms.
  8. Let the worms settle to the bottom of the tubes - about 10 minutes
  9. Remove all but 1 ml of the water with a disposable Pasteur pipette.
  10. Add sterile water to 15 ml and cap the tube again. Invert and let the worms settle.
  11. Remove all but 1 ml of the water with a disposable Pasteur pipette.
  12. Transfer the remaining 1 ml of water + worms to a sterile 1.5 ml microfuge tube.
  13. Spin the worms at 10,000 rpm for 1 minute to pellet.
  14. Remove all but approximately 50 ul of worms and water at the bottom of the tube.
  15. Using a razor blade, cut the end off of a micropipette tip (this will prevent the pellet of worms from being damaged).
  16. Pipette the entire pellet of worms to the appropriate assay plate on the central 2 cm circle.
  17. Wick away any extra water with the corner of a Kimwipe

While the C. elegans are settling: Add 3 μL of 0.25M sodium azide next to the Na and W dots on your chemotaxis assay plates. The sodium azide will immobilize the worms close to the dots

The Assay:

  1. The worms are allowed to move around each plate for 1 hour
  2. Take a photograph of each set of plates (rrf-3 from RNAi Feeder and HT115(DE) control plates; N2 from RNAi Feeder and HT115(DE) control plates; and lsy-2 mutants) at the end of the hour using the Bio-Rad imager in the Instrument Room L308. (Directions are found below.) Make sure you carefully organize the 3 plates for the rrf-3 and Ns strains so that the photo of the rrf-3 worm strain has the worms fed dsRNA of lsy-2 and no lsy-2(control) in the same comparative position as the photo of the 3 N2 worm strain plates. There are only be 2 chemotaxis plates of a lsy-2 mutant (replicates) in that set so you can add a photo of the chemotaxis control that got no NaCl (water on both dots).
  3. At the end of the hour after photographing the plates, the worms in the two quandrants nearest the Na dot are counted and compared to the number in the two quandrants near the W dot. Counting is best done by inverting the plates and using your dissecting scope and a sharpie making a dot on the plastic surface of the plate for every worm you see. Then dots/quandrant can be counted and recorded and general trends in the worm distribution can be observed and notes recorded in your lab notebook.
  4. With a different color sharpie for each strain circle the area of the plate containing the greatest concentration of worms - generally this shape will be a circle or oval.
  5. Take a second set of photographs, arranging the plates and quandrants in exactly the same positions as in the first set of photos.

Capturing Digital Images of Nucleic Acid Gels Stained with SYBR Safe Using the BioRad Imaging System in L308

Instructions for Taking a DNA gel image stained with Sybr Safe using the BioRad ChemiDoc MP System with Image Lab Software

IMPORTANT: Ethidium Bromide stained gels may NOT be imaged in this equipment. Remove gloves and wash hands BEFORE using the computer. DO NOT contaminate the computer. The XcitaBLue Conversion Screen should be kept covering the UV transilluminator and only removed TEMPORARILY when using fluorphores other than Sybr Safe or SybrGreen. Please return the Xcita Blue Screen to the UV transillumintor if you remove it.

Quick and Easy Protocol for photographing your chemotaxis experiment:
1) Make sure the Power Button on the right front of the imager shows a green light. If not press it until the green light comes on and wait 5-10 min for warm up.

2) Open the UV transilluminator drawer on the lower front of the imager and remove the XcitaBlue Conversion Screen. Place it carefully in a save area and don't forget to return it to where you found it when you are done.

3) Position your plates in the center of the drawer.

4) Close the drawer.

5) Open the ImageLab 4.0.1 software by double clicking on the icon on the computer desktop

6) Find and open the Protocol called ????. Double click to open it.

7) Click Position (yellow button) to check the position of your plates.

8) Click Run Protocol (green button) to take the photo.

9) To Save your image, find or make a folder for your work in Documents (NOT on the Desktop). go to File---Export---Export for Publication (use the defaults, e.g. 300dpi). You will see a pop up message reminding you that this option is not optimized for analysis---it doesn’t matter—Click OK. Check the Location where your image will be saved, the FILE Name, and use the drop down menu to SAVE AS TYPE tiff or jpg. Click Save.

10) Close the Image Lab Software 11) Remove your plates 12) The computer AND the ChemiDoc Imager should remain ON. DO NOT Turn OFF the power or shut down or log off the computer.

Links to Labs& Project Info

Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Continued
Lab 6: DNA sequence analysis; Mapping Continued
Lab 7: Complete Mapping: Score
Background Information on Project 3: Investigating Gene Regulation Using RNAi
Media Recipes
Lab 7: Identifying a bacterial colony containing our plasmid of interest
Lab 8: Creating the feeding strain of bacteria for RNAi
Lab 9: Induction of feeding strain to produce dsRNA and feeding worms
Lab 10: Phenotypic analysis of treated vs untreated worms
Lab 11: Writing Workshop
Lab 12: Writing Conferences