DNase Protocol - Revision history

Torsten Waldminghaus: add back to protocol link

2009-02-23T16:34:48Z

add back to protocol link

←Older revision		Revision as of 16:34, 23 February 2009
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		+	{{back to protocols}}
		+
	==Overview==		==Overview==

Torsten Waldminghaus at 14:42, 3 February 2009

2009-02-03T14:42:56Z

←Older revision		Revision as of 14:42, 3 February 2009
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	==Materials==		==Materials==
		+	*The DNAfree kit contains the stuff you need: [https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid=2LdHJLWLKLylLhcGty6LnMnXzP2qcg66GJJ0XTL7s2zKXPFyqwBN!1659201712?cmd=catNavigate2&catID=603181 Applied Biosystems]
	*THAWED 10X DNase Buffer		*THAWED 10X DNase Buffer

Torsten Waldminghaus: /* Notes */

2009-02-03T14:24:40Z

Notes

←Older revision		Revision as of 14:24, 3 February 2009
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	==Notes==		==Notes==
-	~~#List troubleshooting tips here.~~
-
	*[[User:Smoore\|Smoore]] 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesium. If you are adding DNase I to degrade DNA in an existing solution (like a lysate), having 0.5-1.0 mM CaCl2 seems to really help. I don't know if it is in the kit buffer.		*[[User:Smoore\|Smoore]] 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesium. If you are adding DNase I to degrade DNA in an existing solution (like a lysate), having 0.5-1.0 mM CaCl2 seems to really help. I don't know if it is in the kit buffer.

	*[[User:Smoore\|Smoore]] 09:59, 3 December 2008 (EST)If you are poor and rely on heating your samples to kill the DNase, make sure to chelate the metals with excess EDTA before heating. Hot + metal = degraded RNA.		*[[User:Smoore\|Smoore]] 09:59, 3 December 2008 (EST)If you are poor and rely on heating your samples to kill the DNase, make sure to chelate the metals with excess EDTA before heating. Hot + metal = degraded RNA.

-		+	Please sign your name to your note by adding <font face="courier"><nowiki>'''~~~~''':</nowiki></font> to the beginning of your tip.
-		+
-	~~#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.~~	+
-		+
-	~~#Anecdotal observations that might be of use to others can also be posted here.~~	+
-		+
-		+
-	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.	+

	==References==		==References==

Torsten Waldminghaus: /* Materials */

2009-02-03T14:22:06Z

Materials

←Older revision		Revision as of 14:22, 3 February 2009
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	==Materials==		==Materials==
	*THAWED 10X DNase Buffer		*THAWED 10X DNase Buffer
-	*
-	*X μL reagent 2
-	**component A (reagent 2 is made up of multiple components)
-	**component B
-	*equipment 1
-	*equipment 2

	==Procedure==		==Procedure==

Smoore: /* Notes */

2008-12-03T14:59:41Z

Notes

←Older revision		Revision as of 14:59, 3 December 2008
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	==Notes==		==Notes==
	#List troubleshooting tips here.		#List troubleshooting tips here.
		+
		+	*[[User:Smoore\|Smoore]] 09:59, 3 December 2008 (EST)DNase I requires calcium as well as magnesium. If you are adding DNase I to degrade DNA in an existing solution (like a lysate), having 0.5-1.0 mM CaCl2 seems to really help. I don't know if it is in the kit buffer.
		+
		+	*[[User:Smoore\|Smoore]] 09:59, 3 December 2008 (EST)If you are poor and rely on heating your samples to kill the DNase, make sure to chelate the metals with excess EDTA before heating. Hot + metal = degraded RNA.
		+
		+
		+
	#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.		#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
		+
	#Anecdotal observations that might be of use to others can also be posted here.		#Anecdotal observations that might be of use to others can also be posted here.
		+

	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

Stevej: /* Procedure */

2007-05-24T15:16:07Z

Procedure

←Older revision		Revision as of 15:16, 24 May 2007
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	==Procedure==		==Procedure==
	#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:		#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
-	::*~~10ul~~ 10X DNase buffer	+	::*10μL 10X DNase buffer
-	::*~~78ul~~ dH<sub>2</sub>0	+	::*78μL dH<sub>2</sub>0
-	::*~~2ul~~ DNase	+	::*2μL DNase
-	::*~~10ul~~ RNA(1ug/ul)	+	::*10μL RNA(1ug/μL)
	#Incubate at 37° C for 20-30 min.		#Incubate at 37° C for 20-30 min.
	#Add 0.1 volume resuspended DNase Inactivation Reagent.		#Add 0.1 volume resuspended DNase Inactivation Reagent.

Stevej: /* Materials */

2007-05-24T15:14:20Z

Materials

←Older revision		Revision as of 15:14, 24 May 2007
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	==Materials==		==Materials==
-	List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.	+	*THAWED 10X DNase Buffer
-		+	*
-	*~~supply 1 (i.e. tubes of a certain size? spreaders?)~~	+
-	*~~reagent 1~~	+
	*X μL reagent 2		*X μL reagent 2
	**component A (reagent 2 is made up of multiple components)		**component A (reagent 2 is made up of multiple components)

Stevej: /* Overview */

2007-05-24T15:12:16Z

Overview

←Older revision		Revision as of 15:12, 24 May 2007
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	==Overview==		==Overview==

-	Protocol for ~~the Ambion DNA-free~~ DNase ~~kit~~. This uses a DNase inactivating reagent and thus avoids the DNase heat inactivation step that makes me nervous.	+	Protocol for DNase treatment of total RNA. This uses a DNase inactivating reagent and thus avoids the DNase heat inactivation step that makes me nervous.

	==Materials==		==Materials==

Stevej: /* Procedure */

2007-05-24T15:11:38Z

Procedure

←Older revision		Revision as of 15:11, 24 May 2007
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	==Procedure==		==Procedure==
-	#In order to DNase 20ug of RNA in a 100ul reaction, ~~mixup~~ the following:	+	#In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
	::*10ul 10X DNase buffer		::*10ul 10X DNase buffer
	::*78ul dH<sub>2</sub>0		::*78ul dH<sub>2</sub>0

Stevej: /* Procedure */

2007-05-24T05:37:22Z

Procedure

←Older revision		Revision as of 05:37, 24 May 2007
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	::*78ul dH<sub>2</sub>0		::*78ul dH<sub>2</sub>0
	::*2ul DNase		::*2ul DNase
-	::10ul RNA(1ug/ul)	+	::*10ul RNA(1ug/ul)
	#Incubate at 37° C for 20-30 min.		#Incubate at 37° C for 20-30 min.
		+	#Add 0.1 volume resuspended DNase Inactivation Reagent.
		+	#Incubate 2 min at RT mixing every 30s.
		+	#Centrifuge at 10,000xg for 1.5 min.
		+	#Transfer solution to a new tube avoiding pelleted Inactivation Reagent.

	==Notes==		==Notes==