Chromosomal DNA isolation from E. coli - Revision history

Daniel Schindler: /* Procedure */

2012-07-31T15:16:52Z

Procedure

←Older revision		Revision as of 15:16, 31 July 2012
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	* incubate 5 min at 65°C		* incubate 5 min at 65°C
	* add 750 μL isopropanol and mix		* add 750 μL isopropanol and mix
-	* spin at max. speed for 5 min	+	* spin at max. speed for 5 min at room temperature and discard supernatant
	* resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml)		* resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml)
	* incubate for 30 min at 65°C		* incubate for 30 min at 65°C

Jerry D King: /* Equipment */

2010-10-27T15:14:58Z

Equipment

←Older revision		Revision as of 15:14, 27 October 2010
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	===Equipment===		===Equipment===
-	*water-bath at 65°C	+	*water bath at 65°C

	==Procedure==		==Procedure==

Jerry D King: /* Materials */

2010-10-27T15:14:40Z

Materials

←Older revision		Revision as of 15:14, 27 October 2010
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	*Isopropanol		*Isopropanol
	*Ethanol		*Ethanol
-	*~~3M Na-acetate~~	+	*3 M NaOAc

	===Reagents===		===Reagents===

Jerry D King: /* Procedure */ typo and spaces between numbers and units

2010-10-27T15:13:51Z

Procedure: typo and spaces between numbers and units

←Older revision		Revision as of 15:13, 27 October 2010
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	* Mix samples directly with ice cold [[Killing Buffer]] in ratio 1:1 and put on ice (samples should be processed as fast as possible)		* Mix samples directly with ice cold [[Killing Buffer]] in ratio 1:1 and put on ice (samples should be processed as fast as possible)
	* Spin down cells 3 min max. speed at 4°C and discard supernatant		* Spin down cells 3 min max. speed at 4°C and discard supernatant
-	* resuspend in ~~300μL~~ [[TE]] and add ~~40μL~~ 10%SDS and ~~3μL~~ 0.5M EDTA	+	* resuspend in 300 μL [[TE]] and add 40 μL 10% SDS and 3 μL 0.5 M EDTA
	* incubate 5 min at 65°C		* incubate 5 min at 65°C
-	* add ~~750μL isopropanole~~ and mix	+	* add 750 μL isopropanol and mix
	* spin at max. speed for 5 min		* spin at max. speed for 5 min
-	* resuspend pellet in ~~500μL~~ TE and add ~~2μL~~ RNase A (~~25mg~~/ml)	+	* resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml)
	* incubate for 30 min at 65°C		* incubate for 30 min at 65°C
-	* add ~~2μL~~ [[proteinase K]] (~~25mg~~/ml) and incubate at 37°C for 15 min	+	* add 2 μL [[proteinase K]] (25 mg/ml) and incubate at 37°C for 15 min
	* phenol extract (2x phenol & 2x chlorophorm)		* phenol extract (2x phenol & 2x chlorophorm)
-	* precipitate over night with ~~1ml~~ ethanol and ~~40μL~~ 3M Na-Acetate	+	* precipitate over night with 1 ml ethanol and 40 μL 3M Na-Acetate
-	* spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in ~~50μL~~ dH<sub>2</sub>O	+	* spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50 μL dH<sub>2</sub>O

	==Critical steps==		==Critical steps==

Michael A. Speer: /* Materials */

2010-07-23T16:47:19Z

Materials

←Older revision		Revision as of 16:47, 23 July 2010
Line 11:		Line 11:
	==Materials==		==Materials==
	*10% SDS		*10% SDS
-	*~~Isopropanole~~	+	*Isopropanol
-	*~~Ethanole~~	+	*Ethanol
	*3M Na-acetate		*3M Na-acetate

Vaishnavi Ananth: /* BioStream version */

2009-11-19T09:53:56Z

BioStream version

←Older revision		Revision as of 09:53, 19 November 2009
Line 47:		Line 47:
	Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>		Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Chromosomal DNA isolation from E. coli protocol in ~~BioStream~~, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	Following is the Chromosomal DNA isolation from E. coli protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
	====Text Output====		====Text Output====
	[[Chromosomal DNA isolation from E. coli protocol]]		[[Chromosomal DNA isolation from E. coli protocol]]

Vaishnavi Ananth at 06:04, 1 October 2009

2009-10-01T06:04:28Z

←Older revision		Revision as of 06:04, 1 October 2009
Line 47:		Line 47:
	Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>		Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>

		+	==BioStream version==
		+	Following is the Chromosomal DNA isolation from E. coli protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
		+	====Text Output====
		+	[[Chromosomal DNA isolation from E. coli protocol]]
		+	====Source Code====
		+	[[Chromosomal DNA isolation from E. coli protocol - source code]]

	[[Category:Protocol]][[Category:Needs attention]]		[[Category:Protocol]][[Category:Needs attention]]

Torsten Waldminghaus: /* Curators */

2009-02-26T16:32:56Z

Curators

←Older revision		Revision as of 16:32, 26 February 2009
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	==Curators==		==Curators==
-	'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] ~~10:01, 9 December 2008 (EST)~~'''	+	'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]]'''

	''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators\|discussing the idea of protocol curators]]. Please contribute.''		''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators\|discussing the idea of protocol curators]]. Please contribute.''

Torsten Waldminghaus: add back to protocol link

2009-02-23T16:32:26Z

add back to protocol link

←Older revision		Revision as of 16:32, 23 February 2009
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		+	{{back to protocols}}
		+
	==Curators==		==Curators==
	'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 10:01, 9 December 2008 (EST)'''		'''[[User:Torsten Waldminghaus\|Torsten Waldminghaus]] 10:01, 9 December 2008 (EST)'''

Torsten Waldminghaus at 11:03, 19 February 2009

2009-02-19T11:03:10Z

←Older revision		Revision as of 11:03, 19 February 2009
Line 14:		Line 14:

	===Reagents===		===Reagents===
-	~~Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.~~	+	*[[TE]]
		+	*[[Killing Buffer]]
		+
	===Equipment===		===Equipment===
-	~~Any equipment used to perform the protocol (link to a method for using them).~~	+	*water-bath at 65°C

	==Procedure==		==Procedure==
-	~~A step by step guide to the experimental procedure.~~	+	* grow culture in your favorite medium
-		+	* Mix samples directly with ice cold [[Killing Buffer]] in ratio 1:1 and put on ice (samples should be processed as fast as possible)
-	~~If you find it helpfull you could use the following icons to highlight important parts:~~	+	* Spin down cells 3 min max. speed at 4°C and discard supernatant
-		+	* resuspend in 300μL [[TE]] and add 40μL 10%SDS and 3μL 0.5M EDTA
-	[[~~Image:Difficult step.png~~]] ~~icon to highlight difficult steps~~	+	* incubate 5 min at 65°C
-		+	* add 750μL isopropanole and mix
-	[[~~Image:Critical step.png~~]] ~~warn about critical steps~~	+	* spin at max. speed for 5 min
-		+	* resuspend pellet in 500μL TE and add 2μL RNase A (25mg/ml)
-	~~[[Image:Time required~~.~~png]] help people plan how long steps will take~~	+	* incubate for 30 min at 65°C
-		+	* add 2μL [[proteinase K]] (25mg/ml) and incubate at 37°C for 15 min
-	[[~~Image:Pause point.png~~]] ~~where protocol can be interrupted~~	+	* phenol extract (2x phenol & 2x chlorophorm)
-		+	* precipitate over night with 1ml ethanol and 40μL 3M Na-Acetate
-	~~[[Image:Optional step.png]] steps that can be omitted or included~~	+	* spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50μL dH<sub>2</sub>O

	==Critical steps==		==Critical steps==
Line 43:		Line 45:
	Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>		Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>

-	~~It might also be good to add an image to show the workflow and timescales for experiment planning.~~
-
-	~~==Acknowledgments==~~
-	~~Acnkowledge any help you had in development, testing, writing this protocol.~~
-
-	~~==References==~~
-	~~See [[OpenWetWare:Biblio]] for information on how to reference within a wiki.~~
-
-	~~==Specific Protocols==~~
-	~~Add links to all the OWW protocols that have been used in making the consensus.~~
-
-	~~==Discussion==~~
-	~~You can [[Talk:{{PAGENAME}}\|discuss this protocol]].~~
-
-	~~Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.~~

-	[[Category:Protocol]]	+	[[Category:Protocol]][[Category:Needs attention]]