In vitro modification of DNA for L. plantarum - Revision history

Michael A. Speer: /* Notes */

Michael A. Speer — Thu, 13 Jan 2011 00:48:03 GMT

Notes

←Older revision		Revision as of 00:48, 13 January 2011
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	*AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.		*AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.
	*There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].		*There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
-	*An alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much ''linear''time, but much less actual time and is much ~~simpler~~. The process goes as follows.	+	*An alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much ''linear'' time, but much less actual time; and is much easier. The process goes as follows.
	::#Miniprep the desired shuttle vector from ''E. coli''.		::#Miniprep the desired shuttle vector from ''E. coli''.
	::#Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.		::#Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.

Michael A. Speer: /* Notes */

Michael A. Speer — Thu, 13 Jan 2011 00:47:02 GMT

Notes

Michael A. Speer: /* Notes */

Michael A. Speer — Thu, 13 Jan 2011 00:44:04 GMT

Notes

←Older revision		Revision as of 00:44, 13 January 2011
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	::*Miniprep the desired shuttle vector from ''E. coli''.		::*Miniprep the desired shuttle vector from ''E. coli''.
	::*Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.		::*Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
-	***Let cells recover in 25ml GM17 media for one hour.	+	::*Let cells recover in 25ml GM17 media for one hour.
-	***Add the appropriate antibiotic to the media.	+	::*Add the appropriate antibiotic to the media.
-	***Grow overnight at 30°C.	+	::*Grow overnight at 30°C.
-	***Miniprep ''L. lactis'' culture.	+	::*Miniprep ''L. lactis'' culture.
-	***Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.	+	::*Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.
-	***Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!	+	::*Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!

	==References==		==References==

Michael A. Speer: /* Notes */

Michael A. Speer — Thu, 13 Jan 2011 00:43:27 GMT

Notes

←Older revision		Revision as of 00:43, 13 January 2011
Line 52:		Line 52:
	#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].		#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
	#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.		#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.
-	***Miniprep the desired shuttle vector from ''E. coli''.	+	::*Miniprep the desired shuttle vector from ''E. coli''.
-	***Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.	+	::*Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
	***Let cells recover in 25ml GM17 media for one hour.		***Let cells recover in 25ml GM17 media for one hour.
	***Add the appropriate antibiotic to the media.		***Add the appropriate antibiotic to the media.

Michael A. Speer: /* Overview */

Michael A. Speer — Fri, 24 Sep 2010 00:58:46 GMT

Overview

←Older revision		Revision as of 00:58, 24 September 2010
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	==Overview==		==Overview==

-	The following is a procedure for the in vitro modification of DNA before electrotransformation into ''Lactobacillus plantarum'' developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including ''Lactobacillus plantarum'' is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium.	+	The following is a procedure for the in vitro modification of DNA before electrotransformation into ''Lactobacillus plantarum'' developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including ''Lactobacillus plantarum'' is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium. See notes for an alternative method.

	==Materials==		==Materials==

Michael A. Speer: /* Notes */

Michael A. Speer — Fri, 24 Sep 2010 00:58:08 GMT

Notes

←Older revision		Revision as of 00:58, 24 September 2010
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	***Miniprep ''L. lactis'' culture.		***Miniprep ''L. lactis'' culture.
	***Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.		***Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.
		+	***Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!

	==References==		==References==

Michael A. Speer: /* Notes */

Michael A. Speer — Fri, 24 Sep 2010 00:57:15 GMT

Notes

←Older revision		Revision as of 00:57, 24 September 2010
Line 52:		Line 52:
	#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].		#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
	#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.		#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.
-	**Miniprep the desired shuttle vector from ''E. coli''.	+	***Miniprep the desired shuttle vector from ''E. coli''.
-	**Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.	+	***Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
-	**Let cells recover in 25ml GM17 media for one hour.	+	***Let cells recover in 25ml GM17 media for one hour.
-	**Add the appropriate antibiotic to the media.	+	***Add the appropriate antibiotic to the media.
-	**Grow overnight at 30°C.	+	***Grow overnight at 30°C.
-	**Miniprep ''L. lactis'' culture.	+	***Miniprep ''L. lactis'' culture.
-	**Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.	+	***Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.

	==References==		==References==

Michael A. Speer: /* Notes */

Michael A. Speer — Fri, 24 Sep 2010 00:56:28 GMT

Notes

←Older revision		Revision as of 00:56, 24 September 2010
Line 51:		Line 51:
	#AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.		#AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.
	#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].		#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
-
	#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.		#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.
	**Miniprep the desired shuttle vector from ''E. coli''.		**Miniprep the desired shuttle vector from ''E. coli''.

Michael A. Speer: /* Notes */

Michael A. Speer — Fri, 24 Sep 2010 00:56:01 GMT

Notes

←Older revision		Revision as of 00:56, 24 September 2010
Line 51:		Line 51:
	#AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.		#AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.
	#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].		#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
		+
		+	#Another alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is way simpler. The process goes as follows.
		+	**Miniprep the desired shuttle vector from ''E. coli''.
		+	**Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
		+	**Let cells recover in 25ml GM17 media for one hour.
		+	**Add the appropriate antibiotic to the media.
		+	**Grow overnight at 30°C.
		+	**Miniprep ''L. lactis'' culture.
		+	**Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.
		+
	==References==		==References==
	#Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)		#Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)

Michael A. Speer: /* DNA Modification */

Michael A. Speer — Tue, 06 Apr 2010 16:11:10 GMT

DNA Modification

←Older revision		Revision as of 16:11, 6 April 2010
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	===DNA Modification===		===DNA Modification===
	1. Add the following to a 25μL aliquot of cell extract:		1. Add the following to a 25μL aliquot of cell extract:
-	** 50μL TNE ~~Solution~~	+	** 50μL TNE Buffer
-	** 10μL of S-adenosylmethionine	+	** 10μL of S-adenosylmethionine Stock Solution
	** 1μL BSA (10mg/ml)		** 1μL BSA (10mg/ml)
	** 10μL of plasmid DNA.		** 10μL of plasmid DNA.

←Older revision		Revision as of 00:47, 13 January 2011
Line 48:		Line 48:
	==Notes==		==Notes==
	All questions, input and feedback are welcome!		All questions, input and feedback are welcome!
-	#AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.	+	*AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
-	#AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.	+	*AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.
-	#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].	+	*There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
-	~~#Another~~ alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much time, but is ~~way~~ simpler. The process goes as follows.	+	*An alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much ''linear''time, but much less actual time and is much simpler. The process goes as follows.
-	::*Miniprep the desired shuttle vector from ''E. coli''.	+	::#Miniprep the desired shuttle vector from ''E. coli''.
-	::*Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.	+	::#Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
-	::*Let cells recover in 25ml GM17 media for one hour.	+	::#Let cells recover in 25ml GM17 media for one hour.
-	::*Add the appropriate antibiotic to the media.	+	::#Add the appropriate antibiotic to the media.
-	::*Grow overnight at 30°C.	+	::#Grow overnight at 30°C.
-	::*Miniprep ''L. lactis'' culture.	+	::#Miniprep ''L. lactis'' culture.
-	::*Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.	+	::#Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.
-	::*Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!	+	::#Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!

	==References==		==References==