Difference between revisions of "Knight:Electroporation"
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Latest revision as of 14:19, 8 May 2007
This protocol is for transforming plasmid DNA into Escherichia coli cells.
For the following, you need one per DNA sample
- Electroporation cuvette (either 1mm or 2mm gap width)
- 1.5 mL eppendorf tube
- LB-agar plate with appropriate antibiotic
- 1mL SOC at room-temperature
- 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.
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.