DEPC - Revision history

Jakob Suckale: /* DEPC-treatment of solutions */

Jakob Suckale — Wed, 15 Apr 2009 13:59:11 GMT

DEPC-treatment of solutions

←Older revision		Revision as of 13:59, 15 April 2009
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	<small>(More DEPC is needed to inactivate larger amounts of RNases, but larger amount of RNases take longer to remove subsequently and increase the risk of residual DEPC, health risk, and interference with downstream applications. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell, whereas 1% is probably the most repeated number.)</small>		<small>(More DEPC is needed to inactivate larger amounts of RNases, but larger amount of RNases take longer to remove subsequently and increase the risk of residual DEPC, health risk, and interference with downstream applications. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell, whereas 1% is probably the most repeated number.)</small>
	*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer		*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer
-	*DEPC must then be completely destroyed by autoclaving (~~15min~~ at 15psi on liquid cycle)	+	*DEPC must then be completely destroyed by autoclaving (15-45min at 15psi on liquid cycle)
		+	<small>Protocols state very different autoclave times. If the fruity smell is faint, autoclaving was sufficient. Even after a long 45min autoclave treatment the smell will not be completely gone. If you use more than 0.05% and have a very sensitive downstream experiment, it does not hurt to autoclave longer or twice.</small>

	==Warnings==		==Warnings==

Jakob Suckale: /* Warnings */ note on smell and illustration

Jakob Suckale — Fri, 30 Jan 2009 16:15:50 GMT

Warnings: note on smell and illustration

←Older revision		Revision as of 16:15, 30 January 2009
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	==Warnings==		==Warnings==
	*DEPC in unstable in aqueous solutions. It hydrolyses quickly to CO<sub>2</sub> and ethanol. Half-life in phosphate buffer at pH 6 is 20min.		*DEPC in unstable in aqueous solutions. It hydrolyses quickly to CO<sub>2</sub> and ethanol. Half-life in phosphate buffer at pH 6 is 20min.
		+
		+	[[Image:Fruits.jpg\|thumb\|DEPC's fruity smell\|right]]

	*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.		*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.

	*Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained. This is especially problematic in centralised water systems with metres of piping.		*Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained. This is especially problematic in centralised water systems with metres of piping.
		+
		+	*The fruity smell coming from ageing DEPC-containing solutions stems from the esters that are generated when ethanol produced from hydrolysis reacts with residual carboxylic esters. It is normal that some fruity smell lingers after DEPC removal and is not necessarily a sign of incomplete DEPC hydrolysis.

	==Effect of residual DEPC on RNA==		==Effect of residual DEPC on RNA==

Jakob Suckale: /* DEPC-treatment of solutions */

Jakob Suckale — Fri, 30 Jan 2009 16:04:25 GMT

DEPC-treatment of solutions

←Older revision		Revision as of 16:04, 30 January 2009
Line 13:		Line 13:
	*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer		*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer
	*DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)		*DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)
-	~~<small>(Esters may be generated during autoclaving giving rise to a 'fruity' smell (that is not coming directly from DEPC).</small>~~

	==Warnings==		==Warnings==

Jakob Suckale: /* DEPC-treatment of solutions */

Jakob Suckale — Fri, 30 Jan 2009 16:03:55 GMT

DEPC-treatment of solutions

←Older revision		Revision as of 16:03, 30 January 2009
Line 10:		Line 10:
	==DEPC-treatment of solutions==		==DEPC-treatment of solutions==
	*add 0.05-0.1% (v/v) DEPC to solution/water		*add 0.05-0.1% (v/v) DEPC to solution/water
-	<small>(~~Many protocols recommend 0.1% but 0.05% works just as well and~~ is ~~easier~~ to remove subsequently. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell)</small>	+	<small>(More DEPC is needed to inactivate larger amounts of RNases, but larger amount of RNases take longer to remove subsequently and increase the risk of residual DEPC, health risk, and interference with downstream applications. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell, whereas 1% is probably the most repeated number.)</small>
	*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer		*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer
	*DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)		*DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)

Jakob Suckale: /* External links */

Jakob Suckale — Thu, 29 Jan 2009 18:25:17 GMT

External links

←Older revision		Revision as of 18:25, 29 January 2009
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	*[http://scienceblogs.com/moleculeoftheday/2007/02/diethyl_pyrocarbonatedepc_maki.php Molecule of the day: DEPC - Making the world safe for RNA everywhere)] ;-)		*[http://scienceblogs.com/moleculeoftheday/2007/02/diethyl_pyrocarbonatedepc_maki.php Molecule of the day: DEPC - Making the world safe for RNA everywhere)] ;-)
	*[http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=3051 DEPC at PubChem]		*[http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=3051 DEPC at PubChem]
		+	*[http://en.wikipedia.org/wiki/Diethylpyrocarbonate DEPC at wikipedia]

	DEPC-related discussion threads:		DEPC-related discussion threads:

Jakob Suckale: /* External links */

Jakob Suckale — Thu, 29 Jan 2009 18:24:35 GMT

External links

←Older revision		Revision as of 18:24, 29 January 2009
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	*[http://www.ambion.com/techlib/tb/tb_178.html RNase and DEPC Treatment: Fact or Laboratory Myth]		*[http://www.ambion.com/techlib/tb/tb_178.html RNase and DEPC Treatment: Fact or Laboratory Myth]
	*[https://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/D5758 Molecular biology grade DEPC from Sigma]		*[https://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/D5758 Molecular biology grade DEPC from Sigma]
-	*[http://scienceblogs.com/moleculeoftheday/2007/02/diethyl_pyrocarbonatedepc_maki.php Molecule of the day: DEPC - Making the world safe for RNA everywhere)] ;-)	+	*[http://scienceblogs.com/moleculeoftheday/2007/02/diethyl_pyrocarbonatedepc_maki.php Molecule of the day: DEPC - Making the world safe for RNA everywhere)] ;-)
		+	*[http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=3051 DEPC at PubChem]

	DEPC-related discussion threads:		DEPC-related discussion threads:

Jakob Suckale: /* DEPC-treatment of solutions */

Jakob Suckale — Thu, 29 Jan 2009 18:19:01 GMT

DEPC-treatment of solutions

←Older revision		Revision as of 18:19, 29 January 2009
Line 10:		Line 10:
	==DEPC-treatment of solutions==		==DEPC-treatment of solutions==
	*add 0.05-0.1% (v/v) DEPC to solution/water		*add 0.05-0.1% (v/v) DEPC to solution/water
-	<small>(Many protocols recommend 0.1% but 0.05% works just as well and is easier to remove subsequently.)</small>	+	<small>(Many protocols recommend 0.1% but 0.05% works just as well and is easier to remove subsequently. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell)</small>
	*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer		*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer
	*DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)		*DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)

Jakob Suckale: /* Warnings */

Jakob Suckale — Thu, 29 Jan 2009 18:13:37 GMT

Warnings

←Older revision		Revision as of 18:13, 29 January 2009
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	*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.		*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.

-	*Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained.	+	*Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained. This is especially problematic in centralised water systems with metres of piping.

	==Effect of residual DEPC on RNA==		==Effect of residual DEPC on RNA==

Jakob Suckale: /* External links */

Jakob Suckale — Thu, 29 Jan 2009 18:12:06 GMT

External links

←Older revision		Revision as of 18:12, 29 January 2009
Line 38:		Line 38:
	*[http://www.ambion.com/techlib/tb/tb_178.html RNase and DEPC Treatment: Fact or Laboratory Myth]		*[http://www.ambion.com/techlib/tb/tb_178.html RNase and DEPC Treatment: Fact or Laboratory Myth]
	*[https://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/D5758 Molecular biology grade DEPC from Sigma]		*[https://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/D5758 Molecular biology grade DEPC from Sigma]
-	*[http://www.ncbi.nlm.nih.gov/pubmed/8643347 Comparison of DEPC-treated water and Milli-Q PF Plus water] (similar RNA degradation in Milli-Q and DEPC-treated water)
	*[http://scienceblogs.com/moleculeoftheday/2007/02/diethyl_pyrocarbonatedepc_maki.php Molecule of the day: DEPC - Making the world safe for RNA everywhere)] ;-)		*[http://scienceblogs.com/moleculeoftheday/2007/02/diethyl_pyrocarbonatedepc_maki.php Molecule of the day: DEPC - Making the world safe for RNA everywhere)] ;-)

Jakob Suckale: /* Warnings */

Jakob Suckale — Thu, 29 Jan 2009 18:11:40 GMT

Warnings

←Older revision		Revision as of 18:11, 29 January 2009
Line 19:		Line 19:

	*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.		*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.
		+
		+	*Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained.

	==Effect of residual DEPC on RNA==		==Effect of residual DEPC on RNA==