Knight:Electroporation

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

Materials
For the following, you need one per DNA sample
 * Electrocompetent cells
 * Plasmid DNA (from a ligation reaction)
 * Ice
 * Ice bucket
 * 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

 * 1) Chill electroporation cuvettes, DNA samples and tubes on ice.
 * 2) Place LB-agar plates in 37&deg;C incubator to warm.
 * 3) Once cuvettes are cold, remove electrocompetent cells from -80&deg;C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.
 * 4) If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled 0.6mL tubes.
 * 5) Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).
 * 6) Dial a P2 pipetman to either 1 or 2&mu;L depending on the salt content of your DNA sample and . Use 2&mu;L for samples that have been purified in some way.
 * 7) Dial a P200 pipetman to 50&mu;L or whatever volume of electrocompetent cells you want to use. Usually 20-50&mu;L.
 * 8) Dial a P1000 pipetman to 950&mu;L and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.
 * 9) Pipet 1-2&mu;L 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.
 * 10) Place cells back on ice to ensure they remain cold.
 * 11) Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold.
 * 12) Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.
 * 13) Wipe off excess moisture from outside of cuvette.
 * 14) Place in chamber of electroporator.
 * 15) Slide the chamber in so that the cuvette sits snugly between electrodes.
 * 16) Pulse the cells with a shock by pressing button on electroporator.
 * 17) Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off.
 * 18) Transfer SOC-cell mixture to chilled eppendorf tube.
 * 19) Chill sample on ice for 2 mins to permit the cells to recover.
 * 20) Transfer eppendorf tube to 37&deg;C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
 * 21) Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200&mu;L but appropriate plating volume depends on efficiency of the transformation.
 * 22) Incubate plate overnight at 37&deg;C.
 * 23) Leave remaining SOC-cell mixture on the benchtop overnight.
 * 24) If you don't have any transformants, plate the rest of the transformation in the morning.