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| 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]] |
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| *[[Electrocompetent Cells | Electrocompetent cells]] | |
| *Plasmid DNA (from a [[Ligation | ligation]] reaction) | |
| *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) | |
| *[[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 promoter 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== |
| | *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]]. |
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| 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]] |
