BISC219/F12: RNAi Lab 10

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.

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 (strain info: OH9016(ot90) is a g to a nucleotide substitution - location not known
6. Wash the worms off of each RNAi feeding plate 3x with ice cold sterile water (kept in your ice bucket) into separately labeled 15ml conical tubes-- store the tubes with worms in them on ice.
7. Start the washing process by pouring 5ml of ice cold sterile water into the bottom of a labeled (worm strain and treatment or control info) conical tube to measure it (does not need to be the exact volume) and then pour that water onto a worm plate. Tilt the plate as you suck up the water and worms into a plastic disposable Pasteur pipet and return the water and worms to the conical tube. Measure another 5 ml of water in a different conical tube and pour it onto the SAME worm plate, tilt, and suck up and the water and worms into the tube with the orginal 5 ml of wash water. Repeat once more until you have 15 ml of water and worms in the labeled tube. Keep the worm tube in the ice bucket! Repeat the collection process for all 8 worm cultures.
8. Let the worms settle to the bottom of the tubes (keep them in the ice bucket!) - about 10 minutes
9. Remove all but ~1 ml of the water with a disposable Pasteur pipette.
10. Pour iced cold sterile water to 15 ml in each tube and cap the tubes again. Invert and let the worms settle.
11. Remove all but ~1 ml of the cold water with a disposable Pasteur pipette.
12. Transfer the remaining 1 ml of cold 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 50μL of worms and water at the bottom of the tube.
15. Using a razor blade, cut about 1/4 inch off the end of a micropipette tip (this will prevent the pellet of worms from being damaged).
16. Pipette the entire pellet of worms and supernatant to the appropriate assay plate, placing the worms gently on the central 2 cm circle.
17. Wick away as much of the water as you can with the corner of a Kimwipe. Do NOT blot! Instead use a one layer corner of the Kimwipe and place it just at the edge of the droplet of water and let the excess water SLOWLY wick into the Kimwipe until the worms are making contact with the medium in the center circle.
18. After you have applied all the C. elegans nematodes to their chemotaxis plates set your timer for one hour.
19. Add 3 μL of 0.25M sodium azide to both the Na and W dots on all of your chemotaxis assay plates. The sodium azide will immobilize the worms who move close to the dots.

The Assay:

1. The worms are allowed to move around each plate for an hour.
2. At the end of the incubation period, count only the worms OUTSIDE THE CENTER CIRCLE by quandrant. You will end up comparing the number of worms within the two quandrants nearest the Na dot (1&2) to the number found in the two quandrants near the W dot (3 &4). Do not count any worms inside the 2cm center circle! You may count worms found on a line between quandrants if you do it systematically, eg. the worm is counted in a quandrant if more than half of its length is in that quandrant. Counting is best done by inverting the plates. Use your dissecting scope and a Sharpie to make a dot on the plastic surface of the plate where every worm you see is located. Then count the dots/quandrant and record the numbers of worms on each quandrant in the google doc data spread sheet in the Project 3 folder in Resources in Sakai. Your instructor will have that spreadsheet open on a computer in the back of the lab. Add the numbers of worms in quandrants 1&2 (Na) and compare that total to the number in quandrants 3&4 (Water). Make conclusions about general trends in the worm distribution and record those notes in your lab notebook.
3. Estimate the number of worms that are left inside the circle (worms NOT counted in the chemotaxis assay quandrants). Record that data in your 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. If there is no particular area with a greater concentration do not make any mark.
5. Take a set of photographs (one per strain of worm: N2, rrf-3 or mutant) using the BIORAD imager (Directions below). Arrange the plates for each strain so that the position of the control plates vs. RNAi treated plates is the same among the photos and that the quandrants are positioned the same in all plates.

Capturing Digital Images 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

Quick and Easy Protocol for photographing your chemotaxis experiment plates:
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 part of the instrument. Find the white opaque tray kept to the left of the computer and put it on top of the UV transilluminator. Position the 3 plates of one strain (N2 or rrf-3 or the 2 plates of the lsy-2 mutant) agar side up on the white tray so that the quandrants in each plate are aligned in the same way.

3) Close the drawer.

4) Open the ImageLab 4.0.1 software (not the Help icon) by double clicking on the icon on the computer desktop

5) Find and open the Recent Protocol called Protocol Chemotaxis 219. Double click to open it.

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

7) When your image appears, check its quality. You can adjust the contrast, etc. using the black and white "sun" icon at the top of the image window.

8) To Save your image, find the BISC219 F12 folder on the Desktop and open it and then open the folder for your lab section. Go to File---Export---Export for Publication (use the defaults, e.g. 300dpi). Check the Location where your image will be saved (make sure it's your lab section's folder), change the FILE Name of the image to the strain name and your team color, and use the drop down menu to SAVE AS TYPE tiff or jpg. Click Save.

9) Remove your plates and repeat with the plates of the next strain. Make sure that you align the plates (RNAi vs. control and quandrant 1 at the top left) exactly as you did the other strain's plates so that the images are easily comparative.

10) Close the Image Lab Software.

11) When all 3 strains have been photographed, Remove your last set of plates.

12) Open Internet Explorer and upload your saved images to the Data folder in Resources in Sakai under Project 3 Chemotaxis Images.

13) If you are the last group to photograph your plates, please remove the white tray and place it back in the rack where you found it. The computer AND the ChemiDoc Imager should remain ON. DO NOT Turn OFF the power or shut down or log off the computer.