Beta-glucuronidase protocols - Revision history

Michael A. Speer: /* Method */

Michael A. Speer — Mon, 30 Jan 2012 05:44:13 GMT

Method

←Older revision		Revision as of 05:44, 30 January 2012
Line 81:		Line 81:
	:6. Resuspend in 750μL GUS buffer.<br>		:6. Resuspend in 750μL GUS buffer.<br>
	:7. Vortex briefly to mix.<br>		:7. Vortex briefly to mix.<br>
-	:8. Incubate for ~~10-15~~ min in 37°C water bath.<br>	+	:8. Incubate for 30 min in 37°C water bath.<br>
	:9. Add 8ul of 10% Triton-X.<br>		:9. Add 8ul of 10% Triton-X.<br>
	:10. Vortex briefly and incubate on ice for 5 mins.<br>		:10. Vortex briefly and incubate on ice for 5 mins.<br>
-	:11. Add ~~75ul~~ of 4-NPG solution and start the timer.<br>	+	:11. Add 80ul of 4-NPG solution and start the timer.<br>
	:12. Incubate in 37°C water bath.<br>		:12. Incubate in 37°C water bath.<br>
	:13. When the color is clearly yellow (between 10 and 30 mins), stop reaction by adding 300μL 1M Na<sub>2</sub>CO<sub>3</sub>		:13. When the color is clearly yellow (between 10 and 30 mins), stop reaction by adding 300μL 1M Na<sub>2</sub>CO<sub>3</sub>

Michael A. Speer: /* Method */

Michael A. Speer — Fri, 27 Jan 2012 04:48:58 GMT

Method

←Older revision		Revision as of 04:48, 27 January 2012
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	===Method===		===Method===
	:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0		:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0
-	:2. Prepare 10mL of Z-Buffer (measures 10 samples) by adding:	+	:2. Prepare 10mL of GUS Buffer (measures 10 samples) by adding:
	::*5mL of sodium phosphate buffer (pH=7)		::*5mL of sodium phosphate buffer (pH=7)
	::*3mL H<sub>2</sub>O		::*3mL H<sub>2</sub>O

Michael A. Speer: /* Assay 2 */

Michael A. Speer — Fri, 27 Jan 2012 04:46:03 GMT

Assay 2

←Older revision		Revision as of 04:46, 27 January 2012
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	*10mM magnesium sulfate solution		*10mM magnesium sulfate solution
	*1M Na<sub>2</sub>CO<sub>3</sub>		*1M Na<sub>2</sub>CO<sub>3</sub>
-	*1mL of 0.1% SDS (w/v in H<sub>2</sub>O)
	*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7)) '''only make 1mL of this!!!'''		*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7)) '''only make 1mL of this!!!'''
	*β-mercaptoethanol		*β-mercaptoethanol

Michael A. Speer: /* Assay 2 */

Michael A. Speer — Thu, 26 Jan 2012 08:25:13 GMT

Assay 2

←Older revision		Revision as of 08:25, 26 January 2012
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	:5. Pellet again by centrifugation.<br>		:5. Pellet again by centrifugation.<br>
	:6. Resuspend in 750μL GUS buffer.<br>		:6. Resuspend in 750μL GUS buffer.<br>
-	:7. Vortex briefly to mix ~~for 10 secs~~.<br>	+	:7. Vortex briefly to mix.<br>
-	:10. Incubate for 10-15 min in 37°C water bath.<br>	+	:8. Incubate for 10-15 min in 37°C water bath.<br>
-	:11. Add ~~50ul~~ of 4-NPG solution and start the timer.<br>	+	:9. Add 8ul of 10% Triton-X.<br>
-	:12. Incubate ~~for 30 minutes~~ in 37°C water bath.<br>	+	:10. Vortex briefly and incubate on ice for 5 mins.<br>
-	:13. ~~After~~ 30 ~~minutes add~~ 300μL 1M Na<sub>2</sub>CO<sub>3</sub> ~~to stop~~ the ~~reaction~~.~~<br>~~	+	:11. Add 75ul of 4-NPG solution and start the timer.<br>
-	:9. Centrifuge the reaction for 1 minute at full speed.<br>	+	:12. Incubate in 37°C water bath.<br>
-	:10. Measure the OD<sub>405</sub> of the supernatant.<br>	+	:13. When the color is clearly yellow (between 10 and 30 mins), stop reaction by adding 300μL 1M Na<sub>2</sub>CO<sub>3</sub>
		+	:14. Record the time.
		+	:15. Centrifuge the reaction for 1 minute at full speed.<br>
		+	:16. Measure the OD<sub>405</sub> of the supernatant.<br>

	===Notes===		===Notes===

Michael A. Speer: /* Assay 2 */

Michael A. Speer — Thu, 26 Jan 2012 08:16:13 GMT

Assay 2

←Older revision		Revision as of 08:16, 26 January 2012
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	*1M Na<sub>2</sub>CO<sub>3</sub>		*1M Na<sub>2</sub>CO<sub>3</sub>
	*1mL of 0.1% SDS (w/v in H<sub>2</sub>O)		*1mL of 0.1% SDS (w/v in H<sub>2</sub>O)
-	*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7))'''(only make 1mL of this)'''	+	*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7)) '''only make 1mL of this!!!'''
	*β-mercaptoethanol		*β-mercaptoethanol
	*10% Triton X-100 (in water)		*10% Triton X-100 (in water)
Line 75:		Line 75:
	::*1mL of magnesium sulfate solution		::*1mL of magnesium sulfate solution
	::*35μL β-mercaptoethanol		::*35μL β-mercaptoethanol
		+	::*20mg Lysozyme

-	:3~~. Measure and record the OD<sub>600</sub> of the cell culture.<br>~~	+	:3. Pellet 1.5 ml of culture by centrifugation for 1 minute.
-	:4. Pellet 1.5 ml of ~~overnight~~ culture by centrifugation for 1 minute.	+	:4. Resuspend in 1ml 100mM sodium phosphate buffer.<br>
-	:5. Resuspend in 1ml ~~gus~~ buffer.<br>	+	:5. Pellet again by centrifugation.<br>
-	:6. Pellet again by centrifugation.<br>	+	:6. Resuspend in 750μL GUS buffer.<br>
-	:7. Resuspend in ~~450μL~~ GUS buffer.<br>	+	:7. Vortex briefly to mix for 10 secs.<br>
-	:~~8. Add 25ul chloroform and 12.5μL 0.1% SDS.<br>~~	+	:10. Incubate for 10-15 min in 37°C water bath.<br>
-	:9. Vortex to mix for 10 secs.<br>	+
-	:10. Incubate for 10 min in 37°C water bath.<br>	+
	:11. Add 50ul of 4-NPG solution and start the timer.<br>		:11. Add 50ul of 4-NPG solution and start the timer.<br>
	:12. Incubate for 30 minutes in 37°C water bath.<br>		:12. Incubate for 30 minutes in 37°C water bath.<br>
-	:13. After 30 minutes add ~~250μL~~ 1M Na<sub>2</sub>CO<sub>3</sub> to stop the reaction.<br>	+	:13. After 30 minutes add 300μL 1M Na<sub>2</sub>CO<sub>3</sub> to stop the reaction.<br>
	:9. Centrifuge the reaction for 1 minute at full speed.<br>		:9. Centrifuge the reaction for 1 minute at full speed.<br>
	:10. Measure the OD<sub>405</sub> of the supernatant.<br>		:10. Measure the OD<sub>405</sub> of the supernatant.<br>

Michael A. Speer: /* Assay 2 */

Michael A. Speer — Thu, 26 Jan 2012 08:08:22 GMT

Assay 2

←Older revision		Revision as of 08:08, 26 January 2012
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	==Assay 2==		==Assay 2==
	===Materials===		===Materials===
		+
		+	*100mM sodium phosphate buffer (pH=7)
		+	:*40mM NaH<sub>2</sub>PO<sub>4</sub>
		+	:*60mM Na<sub>2</sub>HPO<sub>4</sub>
		+	*0.1M potassium chloride solution
		+	*10mM magnesium sulfate solution
	*1M Na<sub>2</sub>CO<sub>3</sub>		*1M Na<sub>2</sub>CO<sub>3</sub>
-	*100mM Sodium Phosphate buffer (pH=7)
-	~~:*50mM NaH<sub>2</sub>PO<sub>4</sub>~~
-	~~:*50mM Na<sub>2</sub>HPO<sub>4</sub>~~
-	*100mM EDTA
	*1mL of 0.1% SDS (w/v in H<sub>2</sub>O)		*1mL of 0.1% SDS (w/v in H<sub>2</sub>O)
-	*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM ~~NaH<sub>2</sub>PO<sub>4</sub>~~)	+	*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7))'''(only make 1mL of this)'''
	*β-mercaptoethanol		*β-mercaptoethanol
-	*~~Chloroform~~	+	*10% Triton X-100 (in water)
-	*Triton X-100	+

	===Method===		===Method===
	:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0		:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0
-	:2. Prepare 10mL of ~~GUS~~ Buffer (measures 10 samples) by adding:	+	:2. Prepare 10mL of Z-Buffer (measures 10 samples) by adding:
-	::*5mL of ~~Sodium Phosphate~~ buffer (pH=7)	+	::*5mL of sodium phosphate buffer (pH=7)
-	::*~~15μL 100mM EDTA~~	+	::*3mL H<sub>2</sub>O
-	::*~~1.5μL Triton X-100~~	+	::*1mL of potassium chloride solution
-	::*~~1μL β-mercaptoethanol~~	+	::*1mL of magnesium sulfate solution
-	::*~~720μL H<sub>2</sub>O~~	+	::*35μL β-mercaptoethanol
		+
	:3. Measure and record the OD<sub>600</sub> of the cell culture.<br>		:3. Measure and record the OD<sub>600</sub> of the cell culture.<br>
	:4. Pellet 1.5 ml of overnight culture by centrifugation for 1 minute.		:4. Pellet 1.5 ml of overnight culture by centrifugation for 1 minute.

Michael A. Speer: /* Method */

Michael A. Speer — Thu, 26 Jan 2012 07:49:45 GMT

Method

←Older revision		Revision as of 07:49, 26 January 2012
Line 68:		Line 68:
	===Method===		===Method===
	:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0		:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0
-	:2. ~~DPrepare~~ 10mL of GUS Buffer (measures 10 samples) by adding:	+	:2. Prepare 10mL of GUS Buffer (measures 10 samples) by adding:
-	::*~~3mL~~ of ~~100mM Na<sub>2</sub>HPO<sub>4</sub>~~	+	::*5mL of Sodium Phosphate buffer (pH=7)
-	~~::*2mL of 100mM NaH<sub>2</sub>PO<sub>4</sub>~~	+
	::*15μL 100mM EDTA		::*15μL 100mM EDTA
	::*1.5μL Triton X-100		::*1.5μL Triton X-100

Michael A. Speer: /* Materials */

Michael A. Speer — Thu, 26 Jan 2012 07:44:46 GMT

Materials

←Older revision		Revision as of 07:44, 26 January 2012
Line 60:		Line 60:
	:*50mM Na<sub>2</sub>HPO<sub>4</sub>		:*50mM Na<sub>2</sub>HPO<sub>4</sub>
	*100mM EDTA		*100mM EDTA
-	*0.1% SDS (w/v in H<sub>2</sub>O)	+	*1mL of 0.1% SDS (w/v in H<sub>2</sub>O)
	*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM NaH<sub>2</sub>PO<sub>4</sub>)		*4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM NaH<sub>2</sub>PO<sub>4</sub>)
	*β-mercaptoethanol		*β-mercaptoethanol

Michael A. Speer: /* Materials */

Michael A. Speer — Thu, 26 Jan 2012 07:42:33 GMT

Materials

←Older revision		Revision as of 07:42, 26 January 2012
Line 58:		Line 58:
	*100mM Sodium Phosphate buffer (pH=7)		*100mM Sodium Phosphate buffer (pH=7)
	:*50mM NaH<sub>2</sub>PO<sub>4</sub>		:*50mM NaH<sub>2</sub>PO<sub>4</sub>
-	:*~~100mM~~ Na<sub>2</sub>HPO<sub>4</sub>	+	:*50mM Na<sub>2</sub>HPO<sub>4</sub>
	*100mM EDTA		*100mM EDTA
	*0.1% SDS (w/v in H<sub>2</sub>O)		*0.1% SDS (w/v in H<sub>2</sub>O)
Line 65:		Line 65:
	*Chloroform		*Chloroform
	*Triton X-100		*Triton X-100
		+
	===Method===		===Method===
	:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0		:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0

Michael A. Speer: /* Assay 2 */

Michael A. Speer — Thu, 26 Jan 2012 07:42:09 GMT

Assay 2

←Older revision		Revision as of 07:42, 26 January 2012
Line 56:		Line 56:
	===Materials===		===Materials===
	*1M Na<sub>2</sub>CO<sub>3</sub>		*1M Na<sub>2</sub>CO<sub>3</sub>
-	*100mM NaH<sub>2</sub>PO<sub>4</sub>	+	*100mM Sodium Phosphate buffer (pH=7)
		+	:*50mM NaH<sub>2</sub>PO<sub>4</sub>
		+	:*100mM Na<sub>2</sub>HPO<sub>4</sub>
	*100mM EDTA		*100mM EDTA
	*0.1% SDS (w/v in H<sub>2</sub>O)		*0.1% SDS (w/v in H<sub>2</sub>O)
Line 64:		Line 66:
	*Triton X-100		*Triton X-100
	===Method===		===Method===
-	:1. ~~Prepare 1500μL~~ of GUS Buffer (~~for each sample to be measured~~) by adding:	+	:1. Grow culture until OD<sub>600</sub> is between 0.6 and 1.0
-	::*~~750μL~~ of 100mM NaH<sub>2</sub>PO<sub>4</sub>	+	:2. DPrepare 10mL of GUS Buffer (measures 10 samples) by adding:
		+	::*3mL of 100mM Na<sub>2</sub>HPO<sub>4</sub>
		+	::*2mL of 100mM NaH<sub>2</sub>PO<sub>4</sub>
	::*15μL 100mM EDTA		::*15μL 100mM EDTA
	::*1.5μL Triton X-100		::*1.5μL Triton X-100