BISC219/F12: RNAi Lab 9 - Revision history

Tucker Crum: /* To Do Today */

Tucker Crum — Wed, 07 Nov 2012 13:17:26 GMT

To Do Today

←Older revision		Revision as of 13:17, 7 November 2012
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	<br>		<br>
	'''To do after induction is complete:'''		'''To do after induction is complete:'''
-	#Pipette 1.5 ml of your induced culture into 4 identical microfuge tubes.	+	#Pipette 1.5 ml (use your P1000 to pipet 750μL twice) of your induced culture into 4 identical microfuge tubes.
	#Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.		#Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.
	#Remove all of the supernatant.		#Remove all of the supernatant.

Tucker Crum: /* To Do Today */

Tucker Crum — Wed, 07 Nov 2012 13:08:00 GMT

To Do Today

←Older revision		Revision as of 13:08, 7 November 2012
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	#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.		#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.
	#Obtain 2 '''control''' plates and label each of them with a '''C '''- these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.		#Obtain 2 '''control''' plates and label each of them with a '''C '''- these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.
-	#At the end of lab today, using a sterilized scalpel, cut 3 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.	+	#At the end of lab today, using a sterilized scalpel, cut 3 small (0.5 cm x 0.5 cm) squares from a starved wild type worm culture.
	#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.		#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.
	#Place the third square onto a control plate.		#Place the third square onto a control plate.
-	#Flame your scalpel blade, cool it for a few seconds and cut ~~two~~ squares from a starved ''rrf-3'' worm culture.	+	#Flame your scalpel blade, cool it for a few seconds and cut 3 similar squares from a starved ''rrf-3'' worm culture.
-	#Place one square onto a pPD129.36+''lsy-2''feeding ~~plate~~ and another square onto a control plate.	+	#Place one square onto each of two pPD129.36+''lsy-2''feeding plates and another square onto a control plate.
	#Incubate all the worms for 7 days at 16°C		#Incubate all the worms for 7 days at 16°C

Melissa Beers: /* To Do Today */

Melissa Beers — Tue, 06 Nov 2012 02:30:04 GMT

To Do Today

←Older revision		Revision as of 02:30, 6 November 2012
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	#Place the third square onto a control plate.		#Place the third square onto a control plate.
	#Flame your scalpel blade, cool it for a few seconds and cut two squares from a starved ''rrf-3'' worm culture.		#Flame your scalpel blade, cool it for a few seconds and cut two squares from a starved ''rrf-3'' worm culture.
-	#Place one square onto a pPD129.38+''lsy-2''feeding plate and another square onto a control plate.	+	#Place one square onto a pPD129.36+''lsy-2''feeding plate and another square onto a control plate.
	#Incubate all the worms for 7 days at 16°C		#Incubate all the worms for 7 days at 16°C

Melissa Beers: /* To Do Today */

Melissa Beers — Tue, 06 Nov 2012 02:29:38 GMT

To Do Today

←Older revision		Revision as of 02:29, 6 November 2012
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	#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.		#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.
	#Obtain 2 '''control''' plates and label each of them with a '''C '''- these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.		#Obtain 2 '''control''' plates and label each of them with a '''C '''- these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.
-	#At the end of lab today, using a sterilized scalpel, cut 4 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.	+	#At the end of lab today, using a sterilized scalpel, cut 3 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.
	#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.		#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.
	#Place the third square onto a control plate.		#Place the third square onto a control plate.
	#Flame your scalpel blade, cool it for a few seconds and cut two squares from a starved ''rrf-3'' worm culture.		#Flame your scalpel blade, cool it for a few seconds and cut two squares from a starved ''rrf-3'' worm culture.
-	#Place one square onto a pPD129.38+''lsy=2''feeding plate and another square onto a control plate.	+	#Place one square onto a pPD129.38+''lsy-2''feeding plate and another square onto a control plate.
	#Incubate all the worms for 7 days at 16°C		#Incubate all the worms for 7 days at 16°C

Tucker Crum: /* Lab 9: Series 3 - Induction of the Feeding Strain to Produce dsRNA and RNAi */

Tucker Crum — Mon, 05 Nov 2012 13:34:22 GMT

Lab 9: Series 3 - Induction of the Feeding Strain to Produce dsRNA and RNAi

←Older revision		Revision as of 13:34, 5 November 2012
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	You and your partner will return to the lab to make an overnight broth culture of your selected colony as described below. This process will create a sub-culture of many identical copies of the bacteria containing the plasmid carrying the construct to RNAi the gene that you want to study.<br>		You and your partner will return to the lab to make an overnight broth culture of your selected colony as described below. This process will create a sub-culture of many identical copies of the bacteria containing the plasmid carrying the construct to RNAi the gene that you want to study.<br>
	<br>		<br>
-	#Find your plate in the glass front refrigerator in a rack labeled with your lab day. Select a colony to start your overnight culture. ~~At this point, ALL of your colonies should contain your~~ plasmid ~~of interest~~. <br>	+	#Find your plate in the glass front refrigerator in a rack labeled with your lab day. Select a large, well isolated colony to start your overnight culture. Do not pick a small "satelite" colony as it may not be transformed with our plasmid. <br>
	#Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.<br>		#Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.<br>
	#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator with the broth) to each tube. '''Calculate the effective concentration of ampicillin''' that you have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>		#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator with the broth) to each tube. '''Calculate the effective concentration of ampicillin''' that you have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>

Tucker Crum: /* To Do Today */

Tucker Crum — Mon, 05 Nov 2012 13:31:37 GMT

To Do Today

←Older revision		Revision as of 13:31, 5 November 2012
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	#Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.		#Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.
	#Remove all of the supernatant.		#Remove all of the supernatant.
-	#Resuspend the bacterial pellets of each tube in 100μL of LB - you are concentrating your bacteria.	+	#Resuspend the bacterial pellets of each tube in 100μL of LB - you are concentrating your bacteria.''
-	#In the laminar flow hood in L304 or L301 (labs next door) pipet all your induced bacteria onto the center of 4 '''feeding plates''' that you have pre-labeled with an F. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin.	+	#In the laminar flow hood in L304 or L301 (labs next door) pipet all your induced bacteria onto the center of 4 '''feeding plates''' that you have pre-labeled with an '''F'''. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin.
	#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.		#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.
-	#Obtain 2 '''control''' plates and ~~labeled~~ them with a C - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.	+	#Obtain 2 '''control''' plates and label each of them with a '''C '''- these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.
	#At the end of lab today, using a sterilized scalpel, cut 4 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.		#At the end of lab today, using a sterilized scalpel, cut 4 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.
	#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.		#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.

Tucker Crum: /* To Do Today */

Tucker Crum — Mon, 05 Nov 2012 13:30:59 GMT

To Do Today

←Older revision		Revision as of 13:30, 5 November 2012
Line 55:		Line 55:
	#Remove all of the supernatant.		#Remove all of the supernatant.
	#Resuspend the bacterial pellets of each tube in 100μL of LB - you are concentrating your bacteria.		#Resuspend the bacterial pellets of each tube in 100μL of LB - you are concentrating your bacteria.
-	#In the laminar flow hood in L304 or L301 (labs next door) pipet all your induced bacteria onto the center of 4 '''feeding plates'''. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin.	+	#In the laminar flow hood in L304 or L301 (labs next door) pipet all your induced bacteria onto the center of 4 '''feeding plates''' that you have pre-labeled with an F. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin.
	#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.		#Allow the bacteria to be absorbed into the medium a bit before you try to move them. Being very careful not to "slosh" the medium around the plate, put your plates in the 37C incubator for ~3 hours.
-	#Obtain 2 '''control''' plates - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.	+	#Obtain 2 '''control''' plates and labeled them with a C - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pPD129.36 plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.
	#At the end of lab today, using a sterilized scalpel, cut 4 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.		#At the end of lab today, using a sterilized scalpel, cut 4 small 0.5 cm x 0.5 cm squares from a starved wild type worm culture.
	#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.		#Using the same scapel, scoop a square and place one square onto a pPD129.36 +''lsy-2'' feeding plate. Repeat to make a replicate.

Tucker Crum: /* Preparation of the Chemotaxis Test Plates */

Tucker Crum — Mon, 05 Nov 2012 13:26:20 GMT

Preparation of the Chemotaxis Test Plates

←Older revision		Revision as of 13:26, 5 November 2012
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	== Preparation of the Chemotaxis Test Plates ==		== Preparation of the Chemotaxis Test Plates ==
-	Obtain from your instructor 8 unseeded salt plates. These will be your chemotaxis assay plates. With the lid on, draw the large and small filled circles on the bottom of the plate exactly as shown. Use the template your instructor will provide and make sure the distances are as the drawing below suggests:	+	Obtain from your instructor 8 unseeded salt plates. These will be your chemotaxis assay plates. With the lid on, draw the large unfilled and small filled circles on the bottom of the plate exactly as shown. Use the template your instructor will provide and make sure the distances are as the drawing below suggests:

	[[Image:Chemotaxis plate diagram.jpg]]		[[Image:Chemotaxis plate diagram.jpg]]

Tucker Crum: /* Preparation of the Chemotaxis Test Plates */

Tucker Crum — Mon, 05 Nov 2012 13:19:42 GMT

Preparation of the Chemotaxis Test Plates

←Older revision		Revision as of 13:19, 5 November 2012
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	== Preparation of the Chemotaxis Test Plates ==		== Preparation of the Chemotaxis Test Plates ==
-	Obtain from your instructor 8 unseeded salt plates. These will be your chemotaxis assay plates. With the lid on draw the large and small filled circles on the bottom of the plate exactly as shown. Use the template your instructor will provide and make sure the distances are as the drawing below suggests:	+	Obtain from your instructor 8 unseeded salt plates. These will be your chemotaxis assay plates. With the lid on, draw the large and small filled circles on the bottom of the plate exactly as shown. Use the template your instructor will provide and make sure the distances are as the drawing below suggests:

	[[Image:Chemotaxis plate diagram.jpg]]		[[Image:Chemotaxis plate diagram.jpg]]

Tucker Crum: /* Preparation of the Chemotaxis Test Plates */

Tucker Crum — Mon, 05 Nov 2012 13:19:08 GMT

Preparation of the Chemotaxis Test Plates

←Older revision		Revision as of 13:19, 5 November 2012
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	== Preparation of the Chemotaxis Test Plates ==		== Preparation of the Chemotaxis Test Plates ==
-	Obtain from your instructor 8 unseeded salt plates. These will be your chemotaxis assay plates. With the lid on ~~label~~ the bottom of the plate as shown:	+	Obtain from your instructor 8 unseeded salt plates. These will be your chemotaxis assay plates. With the lid on draw the large and small filled circles on the bottom of the plate exactly as shown. Use the template your instructor will provide and make sure the distances are as the drawing below suggests:

	[[Image:Chemotaxis plate diagram.jpg]]		[[Image:Chemotaxis plate diagram.jpg]]