Knight:Beta-galactosidase assay/96 well format - Revision history

Austin J. Che at 21:13, 7 April 2008

Austin J. Che — Mon, 07 Apr 2008 21:13:21 GMT

←Older revision		Revision as of 21:13, 7 April 2008
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	*1M NaH<sub>2</sub>PO<sub>4</sub>		*1M NaH<sub>2</sub>PO<sub>4</sub>
	*o-nitrophenyl-β-D-Galactoside ONPG (solid)		*o-nitrophenyl-β-D-Galactoside ONPG (solid)
-	*1M sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>)

	See [[Talk:Knight:Beta-galacosidase assay]] for stock solution recipes.		See [[Talk:Knight:Beta-galacosidase assay]] for stock solution recipes.

Reshma P. Shetty: /* Materials */

Reshma P. Shetty — Thu, 07 Feb 2008 00:50:58 GMT

Materials

←Older revision		Revision as of 00:50, 7 February 2008
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	*1% hexadecyltrimethylammonium bromide (CTAB)		*1% hexadecyltrimethylammonium bromide (CTAB)
	*1% sodium deoxcholate (light-sensitive, stored at 4°C)		*1% sodium deoxcholate (light-sensitive, stored at 4°C)
		+	**10% seems to go funky over time
	*1M NaH<sub>2</sub>PO<sub>4</sub>		*1M NaH<sub>2</sub>PO<sub>4</sub>
	*o-nitrophenyl-β-D-Galactoside ONPG (solid)		*o-nitrophenyl-β-D-Galactoside ONPG (solid)

Reshma P. Shetty: /* Materials */

Reshma P. Shetty — Thu, 07 Feb 2008 00:50:01 GMT

Materials

←Older revision		Revision as of 00:50, 7 February 2008
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	*1M magnesium sulfate (MgSO<sub>4</sub>)		*1M magnesium sulfate (MgSO<sub>4</sub>)
	*1% hexadecyltrimethylammonium bromide (CTAB)		*1% hexadecyltrimethylammonium bromide (CTAB)
-	*10% sodium deoxcholate (light-sensitive, stored at 4°C)	+	*1% sodium deoxcholate (light-sensitive, stored at 4°C)
	*1M NaH<sub>2</sub>PO<sub>4</sub>		*1M NaH<sub>2</sub>PO<sub>4</sub>
	*o-nitrophenyl-β-D-Galactoside ONPG (solid)		*o-nitrophenyl-β-D-Galactoside ONPG (solid)
Line 27:		Line 27:
	*16 μL 1M MgSO<sub>4</sub>		*16 μL 1M MgSO<sub>4</sub>
	*480 μL 1% CTAB		*480 μL 1% CTAB
-	*32 μL 10% sodium deoxycholate	+	*320 μL 1% sodium deoxycholate
	*43.2 μL TCEP (it is a more stable reducing agent than β-mercaptoethanol)		*43.2 μL TCEP (it is a more stable reducing agent than β-mercaptoethanol)
	H<sub>2</sub>O to 8mL		H<sub>2</sub>O to 8mL

Reshma P. Shetty at 17:58, 18 January 2008

Reshma P. Shetty — Fri, 18 Jan 2008 17:58:03 GMT

←Older revision		Revision as of 17:58, 18 January 2008
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	This protocol is an attempt to modify the protocol to 96 well format for assaying many samples in parallel.		This protocol is an attempt to modify the protocol to 96 well format for assaying many samples in parallel.
-
-	~~<font color=red>It is a work in progress and has not been tested!!!</font>~~

	==Materials==		==Materials==
Line 43:		Line 41:

	==Protocol==		==Protocol==
		+
		+	[[Image:Knight96wellbetagalatosidaseassay.JPG\|thumb\|right\|200px\|96 well plate after completion of this assay.]]
		+
	#Grow cultures in tubes under whatever conditions you wish to test.		#Grow cultures in tubes under whatever conditions you wish to test.
	#*96 well plates did not give me as good of growth as tubes.		#*96 well plates did not give me as good of growth as tubes.

Reshma P. Shetty: /* Protocol */

Reshma P. Shetty — Thu, 10 Jan 2008 03:33:19 GMT

Protocol

←Older revision		Revision as of 03:33, 10 January 2008
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	#Grow cultures in tubes under whatever conditions you wish to test.		#Grow cultures in tubes under whatever conditions you wish to test.
	#*96 well plates did not give me as good of growth as tubes.		#*96 well plates did not give me as good of growth as tubes.
-	#*If growing in 96 well ~~plats~~, use incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.	+	#*If growing in 96 well plates, use incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.
	#During growth		#During growth
	##Make permeabilization solution.		##Make permeabilization solution.

Reshma P. Shetty: /* Protocol */

Reshma P. Shetty — Tue, 27 Nov 2007 19:02:15 GMT

Protocol

←Older revision		Revision as of 19:02, 27 November 2007
Line 43:		Line 43:

	==Protocol==		==Protocol==
-	#Grow cultures in ~~a 96 well deep-well plate~~ under whatever conditions you wish to test.	+	#Grow cultures in tubes under whatever conditions you wish to test.
-	#*~~Use~~ incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.	+	#*96 well plates did not give me as good of growth as tubes.
		+	#*If growing in 96 well plats, use incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.
	#During growth		#During growth
	##Make permeabilization solution.		##Make permeabilization solution.
-	##Pre-measure 20 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation.	+	##Pre-measure 80 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation (permeabilization plate).
	#Aliquot cultures into a 96 well microplate (175 μL per well).		#Aliquot cultures into a 96 well microplate (175 μL per well).
-	#Measure Abs<sub>600</sub> of cultures using plate reader.	+	#Measure Abs<sub>600</sub> of cultures using plate reader (absorbance plate).
-	#Remove a 5 μL aliquot of the ~~culture~~ and add it to the ~~20 μL~~ of permeabilization ~~solution~~.	+	#Remove a 20 μL aliquot of each well of the absorbance plate and add it to the corresponding well of the permeabilization plate.
	#*The sample is now stable for several hours. This allows you to perform time-course experiments.		#*The sample is now stable for several hours. This allows you to perform time-course experiments.
	#*Also include a blank (solutions-only) sample for subtracting the background absorbance later.		#*Also include a blank (solutions-only) sample for subtracting the background absorbance later.
-	#~~Make~~ substrate solution.	+	#Once the time course is nearly complete, make substrate solution.
-	~~#Warm samples and substrate solution to 30°C~~	+	#Add 150 μL substrate solution to each well of measurement plate.
-	#Add 150 μL substrate solution to each well.	+	#Add 25 μl of permeabilized samples to measurement plate.
	#Place the plate in the plate reader to measure the A<sub>420</sub> over 60-90 mins.		#Place the plate in the plate reader to measure the A<sub>420</sub> over 60-90 mins.
	#*The plate reader does not actually have a 420 excitation filter. So you must use the CFP 430 excitation filter.		#*The plate reader does not actually have a 420 excitation filter. So you must use the CFP 430 excitation filter.

Reshma P. Shetty: /* Substrate solution */

Reshma P. Shetty — Tue, 27 Nov 2007 18:57:32 GMT

Substrate solution

←Older revision		Revision as of 18:57, 27 November 2007
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	===Substrate solution===		===Substrate solution===
-	For ~~10mL~~	+	For 15mL
-	*1.~~2mL~~ 500mM Na<sub>2</sub>HPO<sub>4</sub>	+	*1.8mL 500mM Na<sub>2</sub>HPO<sub>4</sub>
-	*~~400~~μL 1M NaH<sub>2</sub>PO<sub>4</sub>	+	*600μL 1M NaH<sub>2</sub>PO<sub>4</sub>
-	*10 mg ONPG	+	*15 mg ONPG
-	*27 μL ~~β-mercaptoethanol~~	+	*40.5 μL TCEP (more stable reducing agent than β-mercaptoethanol)
-	**maybe should try substituting TCEP ~~for~~ β-mercaptoethanol ~~since it is a more stable reducing agent~~	+	(You need 150 μL per sample. This is enough for a 96 well plate.)
-		+
-	(You need 150 μL per sample.)	+

	==Protocol==		==Protocol==

Reshma P. Shetty: /* Permeabilization Solution */

Reshma P. Shetty — Tue, 27 Nov 2007 18:56:24 GMT

Permeabilization Solution

←Older revision		Revision as of 18:56, 27 November 2007
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	*480 μL 1% CTAB		*480 μL 1% CTAB
	*32 μL 10% sodium deoxycholate		*32 μL 10% sodium deoxycholate
-	*43.2 μL ~~β-mercaptoethanol~~	+	*43.2 μL TCEP (it is a more stable reducing agent than β-mercaptoethanol)
-	**maybe should try substituting TCEP ~~for β-mercaptoethanol since~~ it is a more stable reducing agent	+
	H<sub>2</sub>O to 8mL		H<sub>2</sub>O to 8mL
	(You need 80 μL per sample. This is enough for a 96 well plate.)		(You need 80 μL per sample. This is enough for a 96 well plate.)

Reshma P. Shetty: /* Permeabilization Solution */

Reshma P. Shetty — Tue, 27 Nov 2007 18:55:37 GMT

Permeabilization Solution

←Older revision		Revision as of 18:55, 27 November 2007
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	===Permeabilization Solution===		===Permeabilization Solution===
-	For ~~2mL~~:	+	For 8mL:
-	*~~400 μL~~ 500 mM Na<sub>2</sub>HPO<sub>4</sub>	+	*1.6 mL 500 mM Na<sub>2</sub>HPO<sub>4</sub>
-	*10μL 4M KCl	+	*40 μL 4M KCl
-	*4μL 1M MgSO<sub>4</sub>	+	*16 μL 1M MgSO<sub>4</sub>
-	*~~160~~μL 1% CTAB	+	*480 μL 1% CTAB
-	*8μL 10% sodium deoxycholate	+	*32 μL 10% sodium deoxycholate
-	*10.8 μL β-mercaptoethanol	+	*43.2 μL β-mercaptoethanol
	**maybe should try substituting TCEP for β-mercaptoethanol since it is a more stable reducing agent		**maybe should try substituting TCEP for β-mercaptoethanol since it is a more stable reducing agent
-	*1407.2 μL H<sub>2</sub>O	+	H<sub>2</sub>O to 8mL
-	(You need 20 μL per sample.)	+	(You need 80 μL per sample. This is enough for a 96 well plate.)

	===Substrate solution===		===Substrate solution===

Reshma P. Shetty: /* Protocol */

Reshma P. Shetty — Tue, 27 Nov 2007 17:48:42 GMT

Protocol

←Older revision		Revision as of 17:48, 27 November 2007
Line 58:		Line 58:
	#Make substrate solution.		#Make substrate solution.
	#Warm samples and substrate solution to 30°C		#Warm samples and substrate solution to 30°C
		+	#Add 150 μL substrate solution to each well.
	#Place the plate in the plate reader to measure the A<sub>420</sub> over 60-90 mins.		#Place the plate in the plate reader to measure the A<sub>420</sub> over 60-90 mins.
	#*The plate reader does not actually have a 420 excitation filter. So you must use the CFP 430 excitation filter.		#*The plate reader does not actually have a 420 excitation filter. So you must use the CFP 430 excitation filter.