Mesoplasma florum:Electroporation - Revision history

Tk: /* Materials */

Tk — Mon, 20 Dec 2010 23:22:12 GMT

Materials

←Older revision		Revision as of 23:22, 20 December 2010
Line 8:		Line 8:
	* chilled mycoplasma electroporation buffer		* chilled mycoplasma electroporation buffer
	** 8 mM HEPES pH 7.4		** 8 mM HEPES pH 7.4
-	** 272 mM sucrose (90.1 g/l)	+	** 272 mM sucrose (93.1 g/l)
	* chilled 1 mm electroporation cuvettes		* chilled 1 mm electroporation cuvettes
	* 10 ml chilled ATCC 1161 medium		* 10 ml chilled ATCC 1161 medium

Tk: /* Materials */

Tk — Mon, 20 Dec 2010 23:21:55 GMT

Materials

←Older revision		Revision as of 23:21, 20 December 2010
Line 8:		Line 8:
	* chilled mycoplasma electroporation buffer		* chilled mycoplasma electroporation buffer
	** 8 mM HEPES pH 7.4		** 8 mM HEPES pH 7.4
-	** 272 mM sucrose	+	** 272 mM sucrose (90.1 g/l)
	* chilled 1 mm electroporation cuvettes		* chilled 1 mm electroporation cuvettes
	* 10 ml chilled ATCC 1161 medium		* 10 ml chilled ATCC 1161 medium

Torsten Waldminghaus at 17:15, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:15:13 GMT

←Older revision		Revision as of 17:15, 23 February 2009
Line 1:		Line 1:
		+	{{back to protocols}}
	Transformation of ''Mesoplasma florum'' by electroporation		Transformation of ''Mesoplasma florum'' by electroporation

-	Materials	+	==Materials==

	* overnight culture of ''Mesoplasma florum'' in ATCC 1161 medium		* overnight culture of ''Mesoplasma florum'' in ATCC 1161 medium
Line 15:		Line 16:
	** Tet selective plates are at 15 μg/ml		** Tet selective plates are at 15 μg/ml

-		+	==Protocol==
-	Protocol	+

	* chill the centrifuge to 4°		* chill the centrifuge to 4°
Line 38:		Line 38:
	* grow plates for 1.5 - 2 days at 30° for colonies		* grow plates for 1.5 - 2 days at 30° for colonies

		+	==Notes==

-	~~Notes~~ 9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt.	+	9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt.
	Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml		Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml


	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 20:59, 13 July 2008

Tk — Sun, 13 Jul 2008 20:59:15 GMT

←Older revision		Revision as of 20:59, 13 July 2008
Line 8:		Line 8:
	** 8 mM HEPES pH 7.4		** 8 mM HEPES pH 7.4
	** 272 mM sucrose		** 272 mM sucrose
-	* chilled 2 mm electroporation cuvettes	+	* chilled 1 mm electroporation cuvettes
-	* chilled ATCC 1161 medium ~~aliquots of 1 ml in 2 ml eppendorfs~~	+	* 10 ml chilled ATCC 1161 medium
	* Selective medium or plates		* Selective medium or plates
	** Tetracycline resistance from TetM is higher than 200 μg/ml		** Tetracycline resistance from TetM is higher than 200 μg/ml
	** Untransformed cells grow poorly at 4 μg/ml		** Untransformed cells grow poorly at 4 μg/ml
-	** ~~Selective~~ plates are at 15 μg/ml	+	** Tet selective plates are at 15 μg/ml


Line 26:		Line 26:
	* add 10 ml of chilled electroporation buffer		* add 10 ml of chilled electroporation buffer
	* spin down again, remove supernatent		* spin down again, remove supernatent
-	* resuspend the pellet in the remaining liquid, bring the total volume to ~~200~~ μl	+	* resuspend the pellet in the remaining liquid, bring the total volume to 1200 μl with EPB
-	* ~~hold the cells on ice~~	+	* Freeze 400 μl aliquots at -80 indefinitely or use immediately
-	* Add 4 μl of transposome or plasmid DNA for transformation	+	* Add 16 μl of transposome or plasmid DNA for transformation
-	* Mix and transfer to chilled 2 mm gap electroporation cuvettes	+	* Mix and transfer to four chilled 1 mm gap electroporation cuvettes
-	* Pulse the cuvette at 2.5 KV	+	* Pulse the cuvette at 1.5 KV
-	* Add ~~200 μl~~ of chilled 1161 medium ~~from a 2 ml tube~~, mix with the pipet	+	* Add ml of chilled 1161 medium immediately, mix with the pipet, cover
-	* Transfer the cuvette contents back into the same tube, ~~repeat~~	+	* Place the cuvettes into the 30° incubator for outgrowth for 50 minutes
-	* Place the ~~2 ml tubes~~ into the 30° incubator for outgrowth for ~~3 hours~~	+
	** cells can be held at 4° following outgrowth		** cells can be held at 4° following outgrowth
	** dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.		** dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.
-	* Plate ~~200~~ μl aliquots on selective plates	+	* Plate 360 μl aliquots on three selective plates
-	* Culture 200 μl in 10 ml selective liquid culture	+	* grow plates for 1.5 - 2 days at 30° for colonies
-	* grow plates ~~and medium~~ at 30° for colonies ~~or color change~~	+

Tk at 19:25, 1 January 2008

Tk — Tue, 01 Jan 2008 19:25:57 GMT

←Older revision		Revision as of 19:25, 1 January 2008
Line 13:		Line 13:
	** Tetracycline resistance from TetM is higher than 200 μg/ml		** Tetracycline resistance from TetM is higher than 200 μg/ml
	** Untransformed cells grow poorly at 4 μg/ml		** Untransformed cells grow poorly at 4 μg/ml
-	** Selective plates are at ~~10 μg/ml or 20~~ μg/ml	+	** Selective plates are at 15 μg/ml

Tk at 01:41, 4 November 2007

Tk — Sun, 04 Nov 2007 01:41:47 GMT

←Older revision		Revision as of 01:41, 4 November 2007
Line 4:		Line 4:

	* overnight culture of ''Mesoplasma florum'' in ATCC 1161 medium		* overnight culture of ''Mesoplasma florum'' in ATCC 1161 medium
		+	** preparing 10 ml cultures with 1, 10, 100, and 1000 μl of infected cultures assures that one of these will be ready the next day at the correct OD level. Select cultures which are just changing color.
	* chilled mycoplasma electroporation buffer		* chilled mycoplasma electroporation buffer
	** 8 mM HEPES pH 7.4		** 8 mM HEPES pH 7.4
	** 272 mM sucrose		** 272 mM sucrose
-	* chilled 1 mm electroporation cuvettes	+	* chilled 2 mm electroporation cuvettes
-	* chilled ATCC 1161 medium aliquots of 10 ml in 15 ml ~~centrifuge tubes~~	+	* chilled ATCC 1161 medium aliquots of 1 ml in 2 ml eppendorfs
	* Selective medium or plates		* Selective medium or plates
	** Tetracycline resistance from TetM is higher than 200 μg/ml		** Tetracycline resistance from TetM is higher than 200 μg/ml
	** Untransformed cells grow poorly at 4 μg/ml		** Untransformed cells grow poorly at 4 μg/ml
-	** Selective plates are at 10 μg/ml	+	** Selective plates are at 10 μg/ml or 20 μg/ml


Line 28:		Line 29:
	* hold the cells on ice		* hold the cells on ice
	* Add 4 μl of transposome or plasmid DNA for transformation		* Add 4 μl of transposome or plasmid DNA for transformation
-	* Mix and transfer to chilled 1 mm gap electroporation cuvettes	+	* Mix and transfer to chilled 2 mm gap electroporation cuvettes
-	* Pulse the cuvette at 1.~~1 to 1.4~~ KV	+	* Pulse the cuvette at 2.5 KV
-	* Add 200 μl of chilled 1161 medium from a 10 ml tube, mix with the pipet	+	* Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet
	* Transfer the cuvette contents back into the same tube, repeat		* Transfer the cuvette contents back into the same tube, repeat
-	* Place the 10 ml tubes into the 30° incubator for outgrowth for 2-3 hours	+	* Place the 2 ml tubes into the 30° incubator for outgrowth for 3 hours
	** cells can be held at 4° following outgrowth		** cells can be held at 4° following outgrowth
-	* ~~spin down cells, discard supernatent~~	+	** dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.
-	* ~~resuspend in~~ the ~~remaining liquid~~, ~~bring to 500~~ μl	+	* Plate 200 μl aliquots on selective plates
-	* Plate ~~100~~ μl aliquots on selective plates	+	* Culture 200 μl in 10 ml selective liquid culture
	* grow plates and medium at 30° for colonies or color change		* grow plates and medium at 30° for colonies or color change

Tk at 18:02, 25 September 2007

Tk — Tue, 25 Sep 2007 18:02:15 GMT

←Older revision		Revision as of 18:02, 25 September 2007
Line 8:		Line 8:
	** 272 mM sucrose		** 272 mM sucrose
	* chilled 1 mm electroporation cuvettes		* chilled 1 mm electroporation cuvettes
-	* chilled ATCC 1161 medium aliquots of 1 ml in 2 ml tubes	+	* chilled ATCC 1161 medium aliquots of 10 ml in 15 ml centrifuge tubes
	* Selective medium or plates		* Selective medium or plates
	** Tetracycline resistance from TetM is higher than 200 μg/ml		** Tetracycline resistance from TetM is higher than 200 μg/ml

Tk at 17:55, 25 September 2007

Tk — Tue, 25 Sep 2007 17:55:05 GMT

←Older revision		Revision as of 17:55, 25 September 2007
Line 23:		Line 23:
	* spin down again, remove supernatent		* spin down again, remove supernatent
	* resuspend the pellet in the remaining liquid		* resuspend the pellet in the remaining liquid
-	* add ~~0.5~~ ml of chilled electroporation buffer, ~~mix~~	+	* add 10 ml of chilled electroporation buffer
		+	* spin down again, remove supernatent
		+	* resuspend the pellet in the remaining liquid, bring the total volume to 200 μl
	* hold the cells on ice		* hold the cells on ice
-	* Aliquot 50 μl of cells into chilled eppendorfs	+	* Add 4 μl of transposome or plasmid DNA for transformation
-	* Add 1 μl of DNA for transformation	+	* Mix and transfer to chilled 1 mm gap electroporation cuvettes
-	* Mix and transfer to a chilled 1 mm gap electroporation ~~cuvette~~	+	* Pulse the cuvette at 1.1 to 1.4 KV
-	* Pulse the cuvette at 1.2 KV	+	* Add 200 μl of chilled 1161 medium from a 10 ml tube, mix with the pipet
-	* Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet	+	* Transfer the cuvette contents back into the same tube, repeat
-	* Transfer the cuvette contents back into the same tube	+	* Place the 10 ml tubes into the 30° incubator for outgrowth for 2-3 hours
-	* Place the 2 ml tubes into the 30° incubator for outgrowth for 2-3 hours	+
	** cells can be held at 4° following outgrowth		** cells can be held at 4° following outgrowth
		+	* spin down cells, discard supernatent
		+	* resuspend in the remaining liquid, bring to 500 μl
	* Plate 100 μl aliquots on selective plates		* Plate 100 μl aliquots on selective plates
-	* Transfer 100 μl aliquots into 10 ml of selective medium
	* grow plates and medium at 30° for colonies or color change		* grow plates and medium at 30° for colonies or color change

Tk at 14:35, 25 September 2007

Tk — Tue, 25 Sep 2007 14:35:06 GMT

Tk at 21:04, 7 September 2007

Tk — Fri, 07 Sep 2007 21:04:11 GMT

←Older revision		Revision as of 21:04, 7 September 2007
Line 37:		Line 37:
	* Transfer 100 μl aliquots into 10 ml of selective medium		* Transfer 100 μl aliquots into 10 ml of selective medium
	* grow plates and medium at 30° for colonies or color change		* grow plates and medium at 30° for colonies or color change
		+
		+
		+	Notes 9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt.
		+	Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml


	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

←Older revision		Revision as of 14:35, 25 September 2007
Line 10:		Line 10:
	* chilled ATCC 1161 medium aliquots of 1 ml in 2 ml tubes		* chilled ATCC 1161 medium aliquots of 1 ml in 2 ml tubes
	* Selective medium or plates		* Selective medium or plates
-	** Tetracycline resistance from TetM is higher than ~~100~~ μg/ml	+	** Tetracycline resistance from TetM is higher than 200 μg/ml
	** Untransformed cells grow poorly at 4 μg/ml		** Untransformed cells grow poorly at 4 μg/ml
	** Selective plates are at 10 μg/ml		** Selective plates are at 10 μg/ml
Line 28:		Line 28:
	* Add 1 μl of DNA for transformation		* Add 1 μl of DNA for transformation
	* Mix and transfer to a chilled 1 mm gap electroporation cuvette		* Mix and transfer to a chilled 1 mm gap electroporation cuvette
-	* Pulse the cuvette at 1.75 KV ~~twice, with a 5 second delay~~	+	* Pulse the cuvette at 1.2 KV
	* Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet		* Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet
	* Transfer the cuvette contents back into the same tube		* Transfer the cuvette contents back into the same tube
-	** transfer may be easier with a gel loading tip to reach into the gap
	* Place the 2 ml tubes into the 30° incubator for outgrowth for 2-3 hours		* Place the 2 ml tubes into the 30° incubator for outgrowth for 2-3 hours
	** cells can be held at 4° following outgrowth		** cells can be held at 4° following outgrowth