Lactobacillus transformation (Speer 2012) - Revision history

Michael A. Speer: /* Notes */

2011-11-18T19:27:21Z

Notes

←Older revision		Revision as of 19:27, 18 November 2011
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	==Notes==		==Notes==
	*All of the liquid reagents/buffers should be autolaved before use.		*All of the liquid reagents/buffers should be autolaved before use.
		+	*Work fast and keep the tubes on ice if you're going to pause for any reason.
	*This protocol works well for chromosomal insertion		*This protocol works well for chromosomal insertion
	*Notice the high amount of DNA added to the competent cells (10%)		*Notice the high amount of DNA added to the competent cells (10%)

2011-11-10T15:12:59Z

Procedure

←Older revision		Revision as of 15:12, 10 November 2011
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	##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock.		##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock.
	##Grow overnight at 30°C without shaking.		##Grow overnight at 30°C without shaking.
-	#'''Make 100ml 2% glycine:'''	+	#'''Make 100ml 2.5% glycine:'''
-	##Add 1g glycine to two flasks of 50ml MRS medium.	+	##Add 1.25g glycine to two flasks of 50ml MRS medium.
	##Shake to dissolve.		##Shake to dissolve.
	#'''Grow cells:'''		#'''Grow cells:'''
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	#'''Treat with ice-cold EDTA:'''		#'''Treat with ice-cold EDTA:'''
	##Centrifuge for 5min at 4000g		##Centrifuge for 5min at 4000g
-	##Resuspend in ~~10ml~~ 50mM EDTA.	+	##Resuspend in 5ml 50mM EDTA.
	##Incubate on ice for 5 minutes		##Incubate on ice for 5 minutes
		+	##Add 25ml ice-cold DI water.
	#'''Wash with ice-cold DI water:'''		#'''Wash with ice-cold DI water:'''
	##Centrifuge culture for 5min at 4000g		##Centrifuge culture for 5min at 4000g

2011-10-06T16:23:05Z

Overview

←Older revision		Revision as of 16:23, 6 October 2011
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	Back to [[Richard Lab:protocols]]<br>		Back to [[Richard Lab:protocols]]<br>
	==Overview==		==Overview==
-	This is basically a combination of the version of the [[Lactobacillus transformation (Berthier 1996)\| Berthier (1996)]] protocol and the Mason (2005)protocol. This protocol is designed for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator. For other protocols check out the [[Electro-transformation of Lactobacillus spp.\|Lactobacillus transformation archive]].	+	This is basically a combination of the version of the [[Lactobacillus transformation (Berthier 1996)\| Berthier (1996)]] protocol and the Mason (2005) protocol. This protocol is designed for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator. For other protocols check out the [[Electro-transformation of Lactobacillus spp.\|Lactobacillus transformation archive]].

	==Materials==		==Materials==

2011-10-06T16:22:54Z

Overview

←Older revision		Revision as of 16:22, 6 October 2011
Line 3:		Line 3:
	Back to [[Richard Lab:protocols]]<br>		Back to [[Richard Lab:protocols]]<br>
	==Overview==		==Overview==
-	This is basically a combination of the version of the [[Lactobacillus transformation (Berthier 1996)\| Berthier (1996) protocol and the Mason (2005)protocol. This protocol is designed for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator. For other protocols check out the [[Electro-transformation of Lactobacillus spp.\|Lactobacillus transformation archive]].	+	This is basically a combination of the version of the [[Lactobacillus transformation (Berthier 1996)\| Berthier (1996)]] protocol and the Mason (2005)protocol. This protocol is designed for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator. For other protocols check out the [[Electro-transformation of Lactobacillus spp.\|Lactobacillus transformation archive]].

	==Materials==		==Materials==

2011-10-06T16:22:39Z

Overview

←Older revision		Revision as of 16:22, 6 October 2011
Line 3:		Line 3:
	Back to [[Richard Lab:protocols]]<br>		Back to [[Richard Lab:protocols]]<br>
	==Overview==		==Overview==
-	This is basically a ~~modified~~ version of the Mason (2005 ~~protocol~~) for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator. For other protocols check out the [[Electro-transformation of Lactobacillus spp.\|Lactobacillus transformation archive]].	+	This is basically a combination of the version of the [[Lactobacillus transformation (Berthier 1996)\| Berthier (1996) protocol and the Mason (2005)protocol. This protocol is designed for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator. For other protocols check out the [[Electro-transformation of Lactobacillus spp.\|Lactobacillus transformation archive]].

	==Materials==		==Materials==

2011-09-16T21:21:25Z

Contact

←Older revision		Revision as of 21:21, 16 September 2011
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	==Contact==		==Contact==
-	*tlr20@psu.edu	+	*Tom Richard (tlr20@psu.edu)
-	*mas853@psu.edu	+	*Mike Speer (mas853@psu.edu)

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

2011-09-16T21:20:55Z

Contact

2011-09-16T21:04:18Z

Notes

←Older revision		Revision as of 21:04, 16 September 2011
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	==Notes==		==Notes==
		+	*All of the liquid reagents/buffers should be autolaved before use.
	*This protocol works well for chromosomal insertion		*This protocol works well for chromosomal insertion
	*Notice the high amount of DNA added to the competent cells (10%)		*Notice the high amount of DNA added to the competent cells (10%)

2011-09-16T21:03:43Z

Materials

←Older revision		Revision as of 21:03, 16 September 2011
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	==Materials==		==Materials==
-	~~All liquids should be autoclaved prior to use.~~
	*MRS media		*MRS media
	*Glycine		*Glycine

2011-09-16T21:03:31Z

Materials

←Older revision		Revision as of 21:03, 16 September 2011
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	==Materials==		==Materials==
		+	All liquids should be autoclaved prior to use.
	*MRS media		*MRS media
	*Glycine		*Glycine