Mesoplasma florum:restriction enzyme tests - Revision history

Torsten Waldminghaus at 17:19, 23 February 2009

2009-02-23T17:19:11Z

←Older revision		Revision as of 17:19, 23 February 2009
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		+	{{back to protocols}}
	Protocol for quick tests of restriction enzyme activity in novel species		Protocol for quick tests of restriction enzyme activity in novel species
	* Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. (1988) Applied Biochemistry and Microbiology (Russia) 24(1):121-124. [http://science.sibenzyme.com/article12_article_14_1.phtml]		* Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. (1988) Applied Biochemistry and Microbiology (Russia) 24(1):121-124. [http://science.sibenzyme.com/article12_article_14_1.phtml]

-		+	==Lysis buffer==
-	Lysis buffer	+
	* 100 mM Tris-HCl pH 8.0		* 100 mM Tris-HCl pH 8.0
	* 50 mM NaCl		* 50 mM NaCl
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	* 0.1% Triton X-100		* 0.1% Triton X-100

-		+	==Reaction buffer==
-	Reaction buffer	+
	* 20 mM Tris-HCl pH 7.5		* 20 mM Tris-HCl pH 7.5
	* 50 mM NaCl		* 50 mM NaCl
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	* This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris		* This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris

-		+	==Procedure==
	* Put a colony or pelleted cells into 250 ul of lysis buffer		* Put a colony or pelleted cells into 250 ul of lysis buffer
	* Incubate 15 minutes at room temperature with shaking or vortexing		* Incubate 15 minutes at room temperature with shaking or vortexing

Tk at 20:11, 5 April 2008

2008-04-05T20:11:28Z

←Older revision		Revision as of 20:11, 5 April 2008
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	* Incubate 1-2 hours at 30°C		* Incubate 1-2 hours at 30°C
	* Run gel		* Run gel
		+
		+
		+	[[category:Mesoplasma florum]][[category:protocol]]

Tk at 19:11, 25 February 2008

2008-02-25T19:11:59Z

←Older revision		Revision as of 19:11, 25 February 2008
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	* 1 mM DTT		* 1 mM DTT
	* This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris		* This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris
		+

	* Put a colony or pelleted cells into 250 ul of lysis buffer		* Put a colony or pelleted cells into 250 ul of lysis buffer

Tk at 19:11, 25 February 2008

2008-02-25T19:11:41Z

←Older revision		Revision as of 19:11, 25 February 2008
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	* 10 mM MgCl<sub>2</sub>		* 10 mM MgCl<sub>2</sub>
	* 1 mM DTT		* 1 mM DTT
-		+	* This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris

	* Put a colony or pelleted cells into 250 ul of lysis buffer		* Put a colony or pelleted cells into 250 ul of lysis buffer

Tk at 19:43, 22 February 2008

2008-02-22T19:43:51Z

←Older revision		Revision as of 19:43, 22 February 2008
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	Lysis buffer		Lysis buffer
-	* 100 mM ~~tris~~-HCl pH 8.0	+	* 100 mM Tris-HCl pH 8.0
	* 50 mM NaCl		* 50 mM NaCl
	* 5 mM EDTA		* 5 mM EDTA
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-	* Put a colony into 250 ul of lysis buffer	+	* Put a colony or pelleted cells into 250 ul of lysis buffer
-	* Incubate 15 minutes at room temperature with shaking	+	* Incubate 15 minutes at room temperature with shaking or vortexing
	* Centrifuge at high speed 1 min		* Centrifuge at high speed 1 min
	* Add 1-4 μL to 20 μL of reaction buffer with 500 ng of lambda DNA		* Add 1-4 μL to 20 μL of reaction buffer with 500 ng of lambda DNA
	* Incubate 1-2 hours at 30°C		* Incubate 1-2 hours at 30°C
	* Run gel		* Run gel

Tk: New page: Protocol for quick tests of restriction enzyme activity in novel species * Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. ...

2008-02-21T23:18:27Z

New page: Protocol for quick tests of restriction enzyme activity in novel species * Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. ...

New page

Protocol for quick tests of restriction enzyme activity in novel species
* Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. (1988) Applied Biochemistry and Microbiology (Russia) 24(1):121-124. [http://science.sibenzyme.com/article12_article_14_1.phtml]

Lysis buffer
* 100 mM tris-HCl pH 8.0
* 50 mM NaCl
* 5 mM EDTA
* 0.1% Triton X-100

Reaction buffer
* 20 mM Tris-HCl pH 7.5
* 50 mM NaCl
* 10 mM MgCl<sub>2</sub>
* 1 mM DTT

* Put a colony into 250 ul of lysis buffer
* Incubate 15 minutes at room temperature with shaking
* Centrifuge at high speed 1 min
* Add 1-4 μL to 20 μL of reaction buffer with 500 ng of lambda DNA
* Incubate 1-2 hours at 30°C
* Run gel