Knight:Electroporation - Revision history

Reshma P. Shetty at 21:19, 8 May 2007

2007-05-08T21:19:35Z

←Older revision		Revision as of 21:19, 8 May 2007
Line 44:		Line 44:

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

Reshma P. Shetty: /* Notes */

2006-12-05T22:41:02Z

Notes

←Older revision		Revision as of 22:41, 5 December 2006
Line 42:		Line 42:
	==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.
-	[[Category:~~E.Coli~~ Protocol]]	+
		+	[[Category:Protocol]]
		+	[[Category:Escherichia coli]]

Jason R. Kelly at 19:53, 18 July 2006

2006-07-18T19:53:42Z

←Older revision		Revision as of 19:53, 18 July 2006
Line 39:		Line 39:
	#Leave remaining SOC-cell mixture on the benchtop overnight.		#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.		#If you don't have any transformants, plate the rest of the transformation in the morning.
		+
		+	==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.
		+	[[Category:E.Coli Protocol]]

Jason R. Kelly: moved to lab specific protocol page b/c updating the ain electroporation page

2006-07-18T19:52:45Z

moved to lab specific protocol page b/c updating the ain electroporation page

New page

This protocol is for transforming plasmid DNA into ''Escherichia coli'' cells.
===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.