Electroporation - Revision history

Michael A. Speer: /* Specific Protocols */

2009-06-23T18:40:53Z

Specific Protocols

←Older revision		Revision as of 18:40, 23 June 2009
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	*[[Knight:Electroporation]]		*[[Knight:Electroporation]]
	*[[Endy:High-efficiency electroporation]]		*[[Endy:High-efficiency electroporation]]
		+	*[[Richard Lab:Electroporation of E. coli]]

	==Notes==		==Notes==

Maximilianh: /* Notes */

2009-06-11T13:42:56Z

Notes

←Older revision		Revision as of 13:42, 11 June 2009
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	==Notes==		==Notes==
-	*If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.	+	*If you are in a hurry and your selection marker is ampicillin and you're sure to have enough plasmid (e.g. not after difficult clonings), you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. This is called [http://www.neb.com/nebecomm/products/protocol120.asp 5-minute transformation] by New England Biolabs and if you follow the link you'll get a nice complete protocol for it (10% efficiency compared to full protocol)
	*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].		*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].

Torsten Waldminghaus at 17:04, 23 February 2009

2009-02-23T17:04:26Z

←Older revision		Revision as of 17:04, 23 February 2009
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		+	{{back to protocols}}
	==Specific Protocols==		==Specific Protocols==
	*[[Knight:Electroporation]]		*[[Knight:Electroporation]]

Reshma P. Shetty at 21:17, 8 May 2007

2007-05-08T21:17:45Z

←Older revision		Revision as of 21:17, 8 May 2007
Line 1:		Line 1:
	==Specific Protocols==		==Specific Protocols==
	*[[Knight:Electroporation]]		*[[Knight:Electroporation]]
-		+	*[[Endy:High-efficiency electroporation]]
-		+

	==Notes==		==Notes==
-
	*If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.		*If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.
-
	*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].		*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].
-
-	*[[Endy:High-efficiency electroporation]]

	[[Category:Protocol]]		[[Category:Protocol]]
		+	[[Category:In vivo]]
	[[Category:Escherichia coli]]		[[Category:Escherichia coli]]

Reshma P. Shetty at 22:41, 5 December 2006

2006-12-05T22:41:39Z

←Older revision		Revision as of 22:41, 5 December 2006
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	*[[Endy:High-efficiency electroporation]]		*[[Endy:High-efficiency electroporation]]

-	[[Category:~~E.Coli~~ Protocol]]	+	[[Category:Protocol]]
		+	[[Category:Escherichia coli]]

Kchang17: /* Notes */

2006-08-09T19:07:08Z

Notes

←Older revision		Revision as of 19:07, 9 August 2006
Line 9:		Line 9:

	*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].		*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].
		+
		+	*[[Endy:High-efficiency electroporation]]

	[[Category:E.Coli Protocol]]		[[Category:E.Coli Protocol]]

Jason R. Kelly at 21:08, 20 July 2006

2006-07-20T21:08:24Z

←Older revision		Revision as of 21:08, 20 July 2006
Line 1:		Line 1:
	==Specific Protocols==		==Specific Protocols==
	*[[Knight:Electroporation]]		*[[Knight:Electroporation]]
		+
		+

	==Notes==		==Notes==

-	If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.	+	*If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.
		+
		+	*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].
		+
	[[Category:E.Coli Protocol]]		[[Category:E.Coli Protocol]]

Jason R. Kelly at 19:52, 18 July 2006

2006-07-18T19:52:21Z

←Older revision		Revision as of 19:52, 18 July 2006
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-	~~This protocol is for transforming plasmid DNA into ''Escherichia coli'' cells.~~	+	==Specific Protocols==
-		+	*[[Knight:Electroporation]]
-	==~~Knight lab~~==	+
-		+
-	~~===Materials===~~	+
-		+
-	*[[~~Electrocompetent Cells \| Electrocompetent cells]]~~	+
-	*Plasmid DNA (from a [[Ligation \| ligation]] reaction)	+
-	*Ice	+
-	*Ice bucket	+
-	~~''For the following, you need one per DNA sample''~~	+
-	*[http:~~//www.btxonline.com/products/cuvettes/~~ Electroporation ~~cuvette~~] ~~(either 1mm or 2mm gap width)~~	+
-	*[[Electroporator]]	+
-	*1.5 mL eppendorf tube	+
-	*LB-agar plate with appropriate antibiotic	+
-	*1mL [[SOC]] at room-temperature	+
-		+
-	~~===Procedure===~~	+
-		+
-	~~#Chill electroporation cuvettes, DNA samples and tubes on ice.~~	+
-	~~#Place LB-agar plates in 37°C incubator to warm.~~	+
-	~~#Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.~~	+
-	~~#If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled 0.6mL tubes.~~	+
-	~~#Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).~~	+
-	~~#Dial a P2 pipetman to either 1 or 2μL depending on the salt content of your DNA sample and . Use 2μL for samples that have been purified in some way.~~	+
-	~~#Dial a P200 pipetman to 50μL or whatever volume of electrocompetent cells you want to use. Usually 20-50μL.~~	+
-	~~#Dial a P1000 pipetman to 950μL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.~~	+
-	~~#Pipet 1-2μL of DNA sample and add to electrocompetent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.~~	+
-	~~#Place cells back on ice to ensure they remain cold.~~	+
-	~~#Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold.~~	+
-	~~#Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.~~	+
-	~~#Wipe off excess moisture from outside of cuvette.~~	+
-	~~#Place in chamber of electroporator.~~	+
-	~~#Slide the chamber in so that the cuvette sits snugly between electrodes.~~	+
-	~~#Pulse the cells with a shock by pressing button on electroporator.~~	+
-	~~#Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off.~~	+
-	~~#Transfer SOC-cell mixture to chilled eppendorf tube.~~	+
-	~~#Chill sample on ice for 2 mins to permit the cells to recover.~~	+
-	~~#Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.~~	+
-	~~#Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200μL but appropriate plating volume depends on efficiency of the transformation.~~	+
-	~~#Incubate plate overnight at 37°C.~~	+
-	~~#Leave remaining SOC-cell mixture on the benchtop overnight.~~	+
-	~~#If you don't have any transformants, plate the rest of the transformation in the morning.~~	+

	==Notes==		==Notes==

ClarkeS: /* Notes */ typo

2006-05-24T22:12:09Z

Notes: typo

←Older revision		Revision as of 22:12, 24 May 2006
Line 45:		Line 45:
	==Notes==		==Notes==

-	If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ~~ampcillin~~ takes a while to be pumped into cells at a high enough concentration to have an effect.	+	If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect.
	[[Category:E.Coli Protocol]]		[[Category:E.Coli Protocol]]

Barry Canton at 17:36, 16 January 2006

2006-01-16T17:36:53Z

←Older revision		Revision as of 17:36, 16 January 2006
Line 46:		Line 46:

	If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampcillin takes a while to be pumped into cells at a high enough concentration to have an effect.		If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampcillin takes a while to be pumped into cells at a high enough concentration to have an effect.
		+	[[Category:E.Coli Protocol]]