Electroporation

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