|
|
| Line 1: |
Line 1: |
| | {{Biotec_Dresden/Header}} | | {{Biotec_Dresden/Header}} |
| - | == Materials ==
| |
| - | * Escherichia coli DH5 alpha electrocompetent cells
| |
| - | * Plasmid DNA
| |
| - | * Ice
| |
| - | * 2mm gap width Electroporation cuvette
| |
| - | * Electroporater
| |
| - | * 1.5ml microfuge tube
| |
| - | * 1mL SOC (room temperature) for each reaction
| |
| - | * LB-agar plate with corresponding antibiotic
| |
| | | | |
| - | == Procedure ==
| + | [[Category:BIOTEC Dresden/Protocol]] |
| - | * 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.
| + | |
| | {{Biotec_Dresden/Bottom}} | | {{Biotec_Dresden/Bottom}} |
"
