Lactobacillus transformation (Berthier 1996) - Revision history

Michael A. Speer: /* Notes */

2011-09-17T01:13:54Z

Notes

←Older revision		Revision as of 01:13, 17 September 2011
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	==Notes==		==Notes==
-	~~All questions, input and feedback~~ are ~~welcome!~~	+	*Time constants for this protocol are between 11.3 and 13.8ms using a Bio-Rad gene pulser with a pulse controller set at 25μF and 600Ω.

	==References==		==References==

2011-09-17T01:11:40Z

Procedure

←Older revision		Revision as of 01:11, 17 September 2011
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	==Procedure==		==Procedure==
-	#Grow cells overnight in MRS Broth at 30°C	+	#Grow cells overnight in MRS Broth at 30°C.
-	#Use overnight culture to inoculate 100ml MRS Broth	+	#Use overnight culture to inoculate 100ml MRS Broth .
-	#Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6	+	#Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6.
		+	#Wash 2x with wash-solution (10mM MgCl<sub>2</sub>)
		+	##Centrifuge for 5min at 3000g.
		+	##Resuspend in 10ml wash-solution.
		+	##Repeat.
		+	#Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
		+	##Centrifuge for 5min at 3,000g
		+	##Resuspend in 10ml E buffer
		+	#Concentrate in Electroporation Buffer
		+	##Centrifuge for 5min at 3,000g
		+	##Resuspend in 500μL E buffer
		+	#Make 50μL aliquots and store at -80°C until use
		+	#Thaw aliquots on ice for use
		+	#Add up to 5μL plasmid DNA
		+	#Electroporate at 9000kV/cm
		+	#Recover cells by adding 500μL of MRS with 80mM MgCl<sub>2</sub>
		+	#Incubate for 2 hours at 30°C
		+	#Plate to select.

	==Notes==		==Notes==

2011-09-17T01:00:36Z

Procedure

←Older revision		Revision as of 01:00, 17 September 2011
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	==Procedure==		==Procedure==
-	#~~Ensure that the [[In vitro modification of DNA for L. plantarum]] protocol has been followed.~~	+	#Grow cells overnight in MRS Broth at 30°C
-	~~#Thaw 50μL of competent~~ cells ~~on ice from aliquots~~ in ~~-80°C freezer.~~	+	#Use overnight culture to inoculate 100ml MRS Broth
-	#~~Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer~~ to ~~50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).~~	+	#Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6
-	#~~Electroporation done~~ at ~~13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.~~	+
-	~~#Add 500 ul of MRS media containing 80mM MgCl~~<sub>2</sub> ~~and 55mM Glucose~~	+
-	~~#Incubate for 2 hours at 30°C~~.	+
-	~~#Plate cells on MRS plates + antibiotic resistance~~.	+

	==Notes==		==Notes==

2011-09-17T00:57:25Z

Materials

←Older revision		Revision as of 00:57, 17 September 2011
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	*Stock solution of MgCl<sub>2</sub>		*Stock solution of MgCl<sub>2</sub>
	*Stock Solution of Glucose		*Stock Solution of Glucose
		+	*Electroporation Buffer (0.5M Sucrose, 10% Glycerol)

	==Procedure==		==Procedure==

2011-09-16T21:36:36Z

Procedure

←Older revision		Revision as of 21:36, 16 September 2011
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	==Procedure==		==Procedure==
	#Ensure that the [[In vitro modification of DNA for L. plantarum]] protocol has been followed.		#Ensure that the [[In vitro modification of DNA for L. plantarum]] protocol has been followed.
-	#Thaw ~~50 ul~~ of competent cells on ice from aliquots in -80°C freezer.	+	#Thaw 50μL of competent cells on ice from aliquots in -80°C freezer.
	#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).		#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).
	#Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.		#Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.

2011-09-16T21:36:13Z

Procedure

←Older revision		Revision as of 21:36, 16 September 2011
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	==Procedure==		==Procedure==
-	#Ensure that the in vitro modification of DNA ~~protocol~~ for ~~''Lactobacillus~~ plantarum'' has been followed.	+	#Ensure that the [[In vitro modification of DNA for L. plantarum]] protocol has been followed.
	#Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.		#Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
	#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).		#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).

2011-04-06T19:09:15Z

←Older revision

Revision as of 19:09, 6 April 2011

2011-04-06T19:08:35Z

←Older revision		Revision as of 19:08, 6 April 2011
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-	Back to [[Protocols]]	+	Back to [[Protocols]]<br>
	Back to [[Electro-transformation of Lactobacillus spp.]]		Back to [[Electro-transformation of Lactobacillus spp.]]
	==Overview==		==Overview==

2011-04-06T19:08:21Z

←Older revision		Revision as of 19:08, 6 April 2011
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		+	Back to [[Protocols]]
		+	Back to [[Electro-transformation of Lactobacillus spp.]]
	==Overview==		==Overview==
	General guidelines for the electro-transformation of ''Lactobacillus sake'' as described by Berthier et al. and as used by Alegre et al. with ''Lactobacillus plantarum''.		General guidelines for the electro-transformation of ''Lactobacillus sake'' as described by Berthier et al. and as used by Alegre et al. with ''Lactobacillus plantarum''.

2011-02-23T21:11:41Z

Materials

←Older revision		Revision as of 21:11, 23 February 2011
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	==Materials==		==Materials==
-	*50 ul of L.plantarum competent cells	+	*50 ul of ''L.plantarum'' competent cells
	*In vitro modified plasmid DNA		*In vitro modified plasmid DNA
	*MRS media + antibiotic		*MRS media + antibiotic