BISC219/F12: RNAi Lab 10 - Revision history

Melissa Beers: /* Lab 10: Series 3 Investigation of Gene Regulation Using RNAi */

2012-11-19T01:42:00Z

Lab 10: Series 3 Investigation of Gene Regulation Using RNAi

←Older revision		Revision as of 01:42, 19 November 2012
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	#Label 1 15 ml conical tubes with '''wild type - control'''		#Label 1 15 ml conical tubes with '''wild type - control'''
	#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 (strain info: OH9016(ot90) is a g to a nucleotide substitution - location not known'''
	#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.		#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.
	#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.		#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.

Tucker Crum: /* Capturing Digital Images Using the BioRad Imaging System in L308 */

2012-11-16T13:59:39Z

Capturing Digital Images Using the BioRad Imaging System in L308

←Older revision		Revision as of 13:59, 16 November 2012
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	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.		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.	+	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.		3) Close the drawer.

Tucker Crum: /* Capturing Digital Images Using the BioRad Imaging System in L308 */

2012-11-16T13:59:25Z

Capturing Digital Images Using the BioRad Imaging System in L308

←Older revision		Revision as of 13:59, 16 November 2012
Line 44:		Line 44:
	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.		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) on the white tray so that the quandrants in each plate are aligned in the same way. Note: Be sure that there is not a lot of condensation on the inside of the plastic top that might interfere with the quality of the image. If there is, use a Kimwipe to remove the condensation before you place the plates on the tray.	+	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.		3) Close the drawer.

Tucker Crum: /* Capturing Digital Images Using the BioRad Imaging System in L308 */

2012-11-16T13:58:26Z

Capturing Digital Images Using the BioRad Imaging System in L308

←Older revision		Revision as of 13:58, 16 November 2012
Line 44:		Line 44:
	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.		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 Position the 3 plates of one strain (N2 or ''rrf-3''or the 2 plates of the ''lsy-2'' mutant) in the ~~drawer~~ so that the quandrants in each plate are aligned in the same way. Note: Be sure that there is no condensation on the inside of the plastic top that ~~will~~ interfere with the quality of the image. If there is, use a Kimwipe to remove the condensation before you place the plates on the ~~glass~~.	+	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) on the white tray so that the quandrants in each plate are aligned in the same way. Note: Be sure that there is not a lot of condensation on the inside of the plastic top that might interfere with the quality of the image. If there is, use a Kimwipe to remove the condensation before you place the plates on the tray.

	3) Close the drawer.		3) Close the drawer.
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	6) Click Run Protocol (green button) to take the photo.		6) Click Run Protocol (green button) to take the photo.

-	7) When your image appears ~~it will have blue stripes across it. Go to the square rainbow icon and open it. Change from Stain Free to Gray and click Ok~~. You can adjust the contrast, etc. ~~by opening~~ the black and white sun icon.	+	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 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 ~~sections~~'), '''change the FILE Name to the strain name and your team color''', and use the drop down menu to SAVE AS TYPE tiff or jpg. Click Save.	+	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.		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	+	10) Close the Image Lab Software.

	11) When all 3 strains have been photographed, Remove your last set of plates.		11) When all 3 strains have been photographed, Remove your last set of plates.
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	12) Open Internet Explorer and upload your saved images to the Data folder in Resources in Sakai under Project 3 Chemotaxis Images.		12) Open Internet Explorer and upload your saved images to the Data folder in Resources in Sakai under Project 3 Chemotaxis Images.

-	13) The computer AND the ChemiDoc Imager should remain ON. DO NOT Turn OFF the power or shut down or log off the computer.	+	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.
	<div class=noprint>		<div class=noprint>

Tucker Crum: /* Lab 10: Series 3 Investigation of Gene Regulation Using RNAi */

2012-11-16T13:51:52Z

Lab 10: Series 3 Investigation of Gene Regulation Using RNAi

Tucker Crum: /* Capturing Digital Images Using the BioRad Imaging System in L308 */

2012-11-15T18:19:00Z

Capturing Digital Images Using the BioRad Imaging System in L308

←Older revision		Revision as of 18:19, 15 November 2012
Line 45:		Line 45:
	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.		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 Position the 3 plates of one strain (N2 or ''rrf-3''or the 2 plates of the ''lsy-2'' mutant) in the drawer so that the quandrants in each plate are aligned in the same way.	+	2) Open the UV transilluminator drawer on the lower front of the imager and Position the 3 plates of one strain (N2 or ''rrf-3''or the 2 plates of the ''lsy-2'' mutant) in the drawer so that the quandrants in each plate are aligned in the same way. Note: Be sure that there is no condensation on the inside of the plastic top that will interfere with the quality of the image. If there is, use a Kimwipe to remove the condensation before you place the plates on the glass.

	3) Close the drawer.		3) Close the drawer.

Tucker Crum: /* Lab 10: Series 3 Investigation of Gene Regulation Using RNAi */

2012-11-15T14:37:15Z

Lab 10: Series 3 Investigation of Gene Regulation Using RNAi

←Older revision		Revision as of 14:37, 15 November 2012
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	#Transfer the remaining 1 ml of cold 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 ~~30μL~~ of worms and water at the bottom of the tube.	+	#Remove all but approximately 30-50μL of worms and water at the bottom of the tube.
	#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).		#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).
	#Pipette the entire pellet of worms and supernatant to the appropriate assay plate, placing the worms gently on the central 2 cm circle.		#Pipette the entire pellet of worms and supernatant to the appropriate assay plate, placing the worms gently on the central 2 cm circle.

Tucker Crum: /* Lab 10: Series 3 Investigation of Gene Regulation Using RNAi */

2012-11-15T14:36:11Z

Lab 10: Series 3 Investigation of Gene Regulation Using RNAi

Tucker Crum: /* Lab 10: Series 3 Investigation of Gene Regulation Using RNAi */

2012-11-14T16:56:01Z

Lab 10: Series 3 Investigation of Gene Regulation Using RNAi

←Older revision		Revision as of 16:56, 14 November 2012
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	#The worms are allowed to move around each plate for 1 hour		#The worms are allowed to move around each plate for 1 hour
	#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.		#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.
-	#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.	+	#At the end of the hour (after photographing the plates), the worms OUTSIDE THE CENTER CIRCLE within the two quandrants nearest the Na dot are counted and compared to the number in the two quandrants near the W dot. Do not count any worms inside the 2cm center circle. You may count worms 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.
	#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.		#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.
	#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.		#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''' ==		=='''Capturing Digital Images Using the BioRad Imaging System in L308''' ==

Tucker Crum: /* Capturing Digital Images Using the BioRad Imaging System in L308 */

2012-11-13T13:27:13Z

Capturing Digital Images Using the BioRad Imaging System in L308

←Older revision		Revision as of 13:27, 13 November 2012
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	'''Instructions for Taking a DNA gel image stained with Sybr Safe using the BioRad ChemiDoc MP System with Image Lab Software'''		'''Instructions for Taking a DNA gel image stained with Sybr Safe using the BioRad ChemiDoc MP System with Image Lab Software'''

-	~~IMPORTANT:~~	+	'''Quick and Easy Protocol for photographing your chemotaxis experiment plates:'''<BR>
-	~~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:'''<BR>	+
	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.		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~~.	+	2) Open the UV transilluminator drawer on the lower front of the imager and Position the 3 plates of one strain (N2 or ''rrf-3''or the 2 plates of the ''lsy-2'' mutant) in the drawer so that the quandrants in each plate are aligned in the same way.

-	3) ~~Position your plates in the center of~~ the drawer.	+	3) Close the drawer.

-	4) ~~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) ~~Open~~ the ~~ImageLab 4~~.0.~~1 software 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) ~~Find and open the~~ Protocol ~~called ????. Double click~~ to ~~open it~~.	+	6) Click Run Protocol (green button) to take the photo.

-	7) ~~Click Position (yellow button)~~ to ~~check~~ the ~~position of your plates~~.	+	7) When your image appears it will have blue stripes across it. Go to the square rainbow icon and open it. Change from Stain Free to Gray and click Ok. You can adjust the contrast, etc. by opening the black and white sun icon.

-	8) ~~Click Run Protocol~~ (~~green button~~) to ~~take~~ the ~~photo~~.	+	8) To Save your image, find the BISC219 F12 folder on the Desktop and open it and 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 sections'), '''change the FILE Name to the strain name and your team color''', and use the drop down menu to SAVE AS TYPE tiff or jpg. Click Save.

-	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~~.	+	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		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.	+	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) The computer AND the ChemiDoc Imager should remain ON. DO NOT Turn OFF the power or shut down or log off the computer.
	<div class=noprint>		<div class=noprint>
		+
	==Links to Labs& Project Info==		==Links to Labs& Project Info==
	'''Series1:'''<BR>		'''Series1:'''<BR>

←Older revision		Revision as of 13:51, 16 November 2012
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	#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 ice cold sterile water (kept in your ice bucket) into separately labeled 15ml conical tubes-- store the tubes with worms in them on ice.		#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.
-	#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 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.	+	#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.
	#Let the worms settle to the bottom of the tubes (keep them in the ice bucket!) - 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~~ iced cold sterile water to 15 ml in each tube and cap the tubes again. Invert and let the worms settle.	+	#Pour 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 cold 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 cold 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 ~~30-~~50μL 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 about 1/4 inch off the end of a micropipette tip (this will prevent the pellet of worms from being damaged).		#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).
	#Pipette the entire pellet of worms and supernatant to the appropriate assay plate, placing the worms gently on the central 2 cm circle.		#Pipette the entire pellet of worms and supernatant to the appropriate assay plate, placing the worms gently on the central 2 cm circle.
	#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.		#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.
-	~~<br>~~	+	#'''After you have applied all the ''C. elegans'' nematodes to their chemotaxis plates set your timer for one hour.'''
-	~~<br>~~	+	#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.
-	'''After you have applied all the ''C. elegans'' ~~cultures~~ to their chemotaxis plates set your timer for ~~the incubation period specified by your instructor:~~''' Add 3 μL of 0.25M sodium azide to both 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:'''
-	#The worms are allowed to move around each plate for ~~a specified period of time~~.	+	#The worms are allowed to move around each plate for an hour.
-	#At the end of the incubation period, the worms OUTSIDE THE CENTER CIRCLE within the two quandrants nearest the Na dot ~~are counted and compared~~ to the number in the two quandrants near the W dot. Do not count any worms inside the 2cm center circle. You may count worms 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.	+	#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.
	# 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.		# 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.
	#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.		#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.

←Older revision		Revision as of 14:36, 15 November 2012
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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) into separately labeled 15ml conical tubes-- store the tubes with worms in them on ice.	+	#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.
-	#~~Once all~~ the ~~worms are collected, put~~ the ~~caps on~~ the ~~tubes~~ and ~~invert~~ a ~~few times~~ to ~~mix~~ the worms.	+	#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 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.
	#Let the worms settle to the bottom of the tubes (keep them in the ice bucket!) - 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 iced cold sterile water to 15 ml in each tube and cap the tubes 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 cold 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 cold 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 ~~50μL~~ of worms and water at the bottom of the tube.	+	#Remove all but approximately 30μL 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 about 1/4 inch off the end 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, placing the worms gently on the central 2 cm circle.	+	#Pipette the entire pellet of worms and supernatant 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 ~~but do not remove your worms~~!	+	#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.
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-	'''~~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.	+	'''After you have applied all the ''C. elegans'' cultures to their chemotaxis plates set your timer for the incubation period specified by your instructor:''' Add 3 μL of 0.25M sodium azide to both 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:'''
-	#The worms are allowed to move around each plate for ~~1 hour~~	+	#The worms are allowed to move around each plate for a specified period of time.
-	~~#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.	+	#At the end of the incubation period, the worms OUTSIDE THE CENTER CIRCLE within the two quandrants nearest the Na dot are counted and compared to the number in the two quandrants near the W dot. Do not count any worms inside the 2cm center circle. You may count worms 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.
-	#At the end of the ~~hour (after photographing the plates)~~, the worms OUTSIDE THE CENTER CIRCLE within the two quandrants nearest the Na dot are counted and compared to the number in the two quandrants near the W dot. Do not count any worms inside the 2cm center circle. You may count worms 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.	+	# 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.
	#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.		#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.
-	#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.	+	#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''' ==		=='''Capturing Digital Images Using the BioRad Imaging System in L308''' ==