Electroporation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(→Materials)
Current revision (14:40, 23 June 2009) (view source)
(→Specific Protocols)
 
(10 intermediate revisions not shown.)
Line 1: Line 1:
-
This protocol is for transforming plasmid DNA into ''Escherichia coli'' cells.
+
{{back to protocols}}
-
 
+
==Specific Protocols==
-
==Knight lab==
+
*[[Knight:Electroporation]]
-
 
+
*[[Endy:High-efficiency electroporation]]
-
===Materials===
+
*[[Richard Lab:Electroporation of E. coli]]
-
 
+
-
*[[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 promoter 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==
 +
*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]].
-
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:Protocol]]
 +
[[Category:In vivo]]
 +
[[Category:Escherichia coli]]

Current revision

back to protocols

Specific Protocols

Notes

  • 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 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 here.
Retrieved from "http://openwetware.org/wiki/Electroporation"
Personal tools