McKinney:TransformByElectroporation - Revision history

Madhadron at 11:47, 14 October 2007

Madhadron — Sun, 14 Oct 2007 11:47:14 GMT

←Older revision		Revision as of 11:47, 14 October 2007
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	[[Category:Protocol]]		[[Category:Protocol]]
-	~~[[Category:In vivo]]~~
	[[Category:Mycobacterium smegmatis]]		[[Category:Mycobacterium smegmatis]]

Madhadron: Transforming mycobacteria by electroporation moved to McKinney:TransformByElectroporation: Moved under the lab where it is used.

Madhadron — Sun, 14 Oct 2007 11:45:33 GMT

Transforming mycobacteria by electroporation moved to McKinney:TransformByElectroporation: Moved under the lab where it is used.

←Older revision

Revision as of 11:45, 14 October 2007

Madhadron: Transformation by electroporation moved to Transforming mycobacteria by electroporation: Added species note

Madhadron — Tue, 25 Sep 2007 14:19:33 GMT

Transformation by electroporation moved to Transforming mycobacteria by electroporation: Added species note

←Older revision

Revision as of 14:19, 25 September 2007

Madhadron at 14:19, 25 September 2007

Madhadron — Tue, 25 Sep 2007 14:19:09 GMT

←Older revision		Revision as of 14:19, 25 September 2007
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	==Notes==		==Notes==

-	Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. ~~tuberculosisi~~'' may transform better at room temperature.	+	Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. tuberculosis'' may transform better at room temperature.

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:In vivo]]		[[Category:In vivo]]
	[[Category:Mycobacterium smegmatis]]		[[Category:Mycobacterium smegmatis]]

Reshma P. Shetty at 19:02, 9 May 2007

Reshma P. Shetty — Wed, 09 May 2007 19:02:11 GMT

←Older revision		Revision as of 19:02, 9 May 2007
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	Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. tuberculosisi'' may transform better at room temperature.		Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. tuberculosisi'' may transform better at room temperature.
		+
		+	[[Category:Protocol]]
		+	[[Category:In vivo]]
		+	[[Category:Mycobacterium smegmatis]]

Madhadron: /* Directions */

Madhadron — Tue, 28 Nov 2006 19:16:58 GMT

Directions

←Older revision		Revision as of 19:16, 28 November 2006
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	# Divide the culture into two 25mL screw cap tubes. Pellet the cells at 3,000rpm for 10 minutes and resuspend each in 25mL glycerol. Repeat twice more, the last time resuspending each in 1mL glycerol. This ensures that the medium is fairly pure. If it's not, then the medium will arc when you electroporate.		# Divide the culture into two 25mL screw cap tubes. Pellet the cells at 3,000rpm for 10 minutes and resuspend each in 25mL glycerol. Repeat twice more, the last time resuspending each in 1mL glycerol. This ensures that the medium is fairly pure. If it's not, then the medium will arc when you electroporate.
	# Prepare the recovery tubes: one test tube of 3mL 7H9 per transformation. Put 300μL of cells in 10% glycerol in each electroporation cuvette, add the DNA (on the order of 3ng per transformation).		# Prepare the recovery tubes: one test tube of 3mL 7H9 per transformation. Put 300μL of cells in 10% glycerol in each electroporation cuvette, add the DNA (on the order of 3ng per transformation).
-	# Electroporate at 2.5kV, 1kΩ, and 50μF. ''Immediately'' transfer the cells to their recovery tube. When all the transformations are done, incubate the recovery tubes shaking at 37°C for at least 3 hours.	+	# Electroporate at 2.5kV, 1kΩ, and 25μF. Should yield time constants of 15 to 25ms. ''Immediately'' transfer the cells to their recovery tube. When all the transformations are done, incubate the recovery tubes shaking at 37°C for at least 3 hours.
	# Pellet the tubes at 3,000 rpm for 10 minutes, and resuspend in 400μL PBS + 0.1% Tween 80. Plate each tube on two LB agar plates with the relevant antibiotics, 200μL per plate. Incubate the plates at 37°C.		# Pellet the tubes at 3,000 rpm for 10 minutes, and resuspend in 400μL PBS + 0.1% Tween 80. Plate each tube on two LB agar plates with the relevant antibiotics, 200μL per plate. Incubate the plates at 37°C.
-

	==Notes==		==Notes==

	Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. tuberculosisi'' may transform better at room temperature.		Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. tuberculosisi'' may transform better at room temperature.

Madhadron at 20:40, 25 September 2006

Madhadron — Mon, 25 Sep 2006 20:40:13 GMT

←Older revision		Revision as of 20:40, 25 September 2006
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	* 50mL 7H9 mycobacterial medium + 3mL per transformation		* 50mL 7H9 mycobacterial medium + 3mL per transformation
	* 102mL 10% glycerol (possibly a few mL more if you are doing many transformations)		* 102mL 10% glycerol (possibly a few mL more if you are doing many transformations)
-	* 400mL PBS + 10% Tween 80 per transformation	+	* 400mL PBS + 0.1% Tween 80 per transformation
	* electroporation cuvettes and electroporator		* electroporation cuvettes and electroporator
	* LB agar plates carrying the relevant antibiotic markers		* LB agar plates carrying the relevant antibiotic markers
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	# Prepare the recovery tubes: one test tube of 3mL 7H9 per transformation. Put 300μL of cells in 10% glycerol in each electroporation cuvette, add the DNA (on the order of 3ng per transformation).		# Prepare the recovery tubes: one test tube of 3mL 7H9 per transformation. Put 300μL of cells in 10% glycerol in each electroporation cuvette, add the DNA (on the order of 3ng per transformation).
	# Electroporate at 2.5kV, 1kΩ, and 50μF. ''Immediately'' transfer the cells to their recovery tube. When all the transformations are done, incubate the recovery tubes shaking at 37°C for at least 3 hours.		# Electroporate at 2.5kV, 1kΩ, and 50μF. ''Immediately'' transfer the cells to their recovery tube. When all the transformations are done, incubate the recovery tubes shaking at 37°C for at least 3 hours.
-	# Pellet the tubes at 3,000 rpm for 10 minutes, and resuspend in 400μL PBS + 10% Tween 80. Plate each tube on two LB agar plates with the relevant antibiotics, 200μL per plate. Incubate the plates at 37°C.	+	# Pellet the tubes at 3,000 rpm for 10 minutes, and resuspend in 400μL PBS + 0.1% Tween 80. Plate each tube on two LB agar plates with the relevant antibiotics, 200μL per plate. Incubate the plates at 37°C.

Madhadron at 19:55, 25 September 2006

Madhadron — Mon, 25 Sep 2006 19:55:10 GMT

New page

==Materials==
* 50mL 7H9 mycobacterial medium + 3mL per transformation
* 102mL 10% glycerol (possibly a few mL more if you are doing many transformations)
* 400mL PBS + 10% Tween 80 per transformation
* electroporation cuvettes and electroporator
* LB agar plates carrying the relevant antibiotic markers
* frozen aliquot of ''M. smegmatis''

==Directions==
# Thaw the aliquot of ''M. smegmatis'' and inoculate 50mL 7H9 with 100-200μL of it (depending on concentration of stock). Incubate shaking at 37°C until the optical density at 600nm is between 0.6 and 0.8. This is the range where you get the highest transformation efficiency.
# Divide the culture into two 25mL screw cap tubes. Pellet the cells at 3,000rpm for 10 minutes and resuspend each in 25mL glycerol. Repeat twice more, the last time resuspending each in 1mL glycerol. This ensures that the medium is fairly pure. If it's not, then the medium will arc when you electroporate.
# Prepare the recovery tubes: one test tube of 3mL 7H9 per transformation. Put 300μL of cells in 10% glycerol in each electroporation cuvette, add the DNA (on the order of 3ng per transformation).
# Electroporate at 2.5kV, 1kΩ, and 50μF. ''Immediately'' transfer the cells to their recovery tube. When all the transformations are done, incubate the recovery tubes shaking at 37°C for at least 3 hours.
# Pellet the tubes at 3,000 rpm for 10 minutes, and resuspend in 400μL PBS + 10% Tween 80. Plate each tube on two LB agar plates with the relevant antibiotics, 200μL per plate. Incubate the plates at 37°C.

==Notes==

Unlike ''E. coli'', there is no need to keep mycobacteria cold when transforming them. Indeed, there is some evidence that ''M. tuberculosisi'' may transform better at room temperature.