BISC219/F12: RNAi Lab 10

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(Lab 10: Series 3 Investigation of Gene Regulation Using RNAi)
(Lab 10: Series 3 Investigation of Gene Regulation Using RNAi)
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#Label 1 15 ml conical tubes with '''''rrf-3'' - control'''
#Label 1 15 ml conical tubes with '''''rrf-3'' - control'''
#Label 2 15 ml conical tubes with '''''lsy-2'' mutant'''
#Label 2 15 ml conical tubes with '''''lsy-2'' mutant'''
-
#Wash the worms off of each RNAi feeding plate 3x with 5 ml ice cold sterile water (kept in your ice bucket)- putting each wash in the correct labeled 15 ml tube - store the tubes with worms in them on ice.
+
#Wash the worms off of each RNAi feeding plate 3x with 5 ml ice cold sterile water (kept in your ice bucket) into separately labeled 15ml conical tubes-- 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.
#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
+
#Let the worms settle to the bottom of the tubes (keep them in the ice bucket!) - about 10  minutes
#Remove all but 1 ml of the water with a disposable Pasteur pipette.
#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.
+
#Add iced cold sterile water to 15 ml in each tube and cap the tubes again.  Invert and let the worms settle.
-
#Remove all but 1 ml of the water with a disposable Pasteur pipette.
+
#Remove all but 1 ml of the cold water with a disposable Pasteur pipette.
-
#Transfer the remaining 1 ml of water + worms to a sterile 1.5 ml microfuge tube.
+
#Transfer the remaining 1 ml of cold water + worms to a sterile 1.5 ml microfuge tube.
#Spin the worms at 10,000 rpm for 1 minute to pellet.   
#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.
+
#Remove all but approximately 50μL 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).
#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 on the central 2 cm circle.
+
#Pipette the entire pellet of worms to the appropriate assay plate, placing the worms gently on the central 2 cm circle.
-
#Wick away any extra water with the corner of a Kimwipe
+
#Wick away any extra water with the corner of a Kimwipe but do not remove your worms!
<br>
<br>
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'''While the ''C. elegans'' are settling:''' Add 3 μL of 0.25M sodium azide on the Na and W dots on your chemotaxis assay plates.  The sodium azide will immobilize the worms close to the dots  
+
'''While the ''C. elegans'' are settling:''' Add 3 μL of 0.25M sodium azide to the Na and W dots on your chemotaxis assay plates.  The sodium azide will immobilize the worms who move close to the dots.
'''The Assay:'''
'''The Assay:'''

Revision as of 08:30, 13 November 2012

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
  6. Wash the worms off of each RNAi feeding plate 3x with 5 ml 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. 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 (keep them in the ice bucket!) - about 10 minutes
  9. Remove all but 1 ml of the water with a disposable Pasteur pipette.
  10. Add 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 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, placing the worms gently on the central 2 cm circle.
  17. Wick away any extra water with the corner of a Kimwipe but do not remove your worms!



While the C. elegans are settling: Add 3 μL of 0.25M sodium azide to the Na and W dots on 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 1 hour
  2. Take a photograph of each of the 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 one of the lab digital cameras or your own camera held to the eye piece of the microscope. Make sure you carefully organize each plate so that the photos have the quadrants in the same comparative position.
  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. 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.
  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 second 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

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

Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
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
Series3:
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

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