"
Team:BIOTEC Dresden/Protocols:Electroporation for ligated products
From 2010.igem.org
(Difference between revisions)
| Line 6: | Line 6: | ||
<h2>Materials</h2> | <h2>Materials</h2> | ||
<ul> | <ul> | ||
| - | + | <li>Escherichia coli DH5 alpha electrocompetent cells</li> | |
| - | + | <li>Plasmid DNA</li> | |
| - | + | <li>Ice</li> | |
| - | + | <li>2mm gap width Electroporation cuvette</li> | |
| - | + | <li>Electroporater</li> | |
| - | + | <li>1.5ml microfuge tube</li> | |
| - | + | <li>1mL SOC (room temperature) for each reaction</li> | |
| - | + | <li>LB-agar plate with corresponding antibiotic</li> | |
</ul> | </ul> | ||
</div> | </div> | ||
| + | |||
<div class="visualClear"></div> | <div class="visualClear"></div> | ||
<div id="procedure"> | <div id="procedure"> | ||
<h2>Procedure</h2> | <h2>Procedure</h2> | ||
<ul> | <ul> | ||
| - | + | <li>Chill electroporation cuvettes, DNA samples and tubes on <span class="temp">ice</span>.</li> | |
| - | + | <li>Place LB-agar plates in <span class="temp">37°C</span> incubator to warm.</li> | |
| - | + | <li>Once cuvettes are cold, remove electrocompetent cells from <span class="temp">-80°C</span> freezer and thaw on <span class="temp">ice</span>. Alternatively, freshly prepared electrocompetent cells may be used immediately.</li> | |
| - | + | <li>If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled <span class="volume">0.6mL</span> tubes.</li> | |
| - | + | <li>Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).</li> | |
| - | + | <li>Dial a P2 pipetman to either <span class="volume">1 or 2μL</span> depending on the salt content of your DNA sample and . Use <span class="volume">2μL</span> for samples that have been purified in some way.</li> | |
| - | + | <li>Dial a P200 pipetman to <span class="volume">50μL</span> or whatever volume of electrocompetent cells you want to use. Usually <span class="volume">20-50μL</span> .</li> | |
| - | + | <li>Dial a P1000 pipetman to <span class="volume">950μL</span> and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.</li> | |
| - | + | <li>Pipet <span class="volume">1-2μL</span> 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.</li> | |
| - | + | <li>Place cells back on <span class="temp">ice</span> to ensure they remain cold.</li> | |
| - | + | <li>Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold.</li> | |
| - | + | <li>Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.</li> | |
| - | + | <li>Wipe off excess moisture from outside of cuvette.</li> | |
| - | + | <li>Place in chamber of electroporator.</li> | |
| - | + | <li>Slide the chamber in so that the cuvette sits snugly between electrodes.</li> | |
| - | + | <li>Pulse the cells with a shock by pressing button on electroporator.</li> | |
| - | + | <li>Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off.</li> | |
| - | + | <li>Transfer SOC-cell mixture to chilled eppendorf tube.</li> | |
| - | + | <li>Chill sample on <span class="temp">ice</span> for <span class="time">2 mins</span> to permit the cells to recover.</li> | |
| - | + | <li>Transfer eppendorf tube to <span class="temp">37°C</span> incubator and shake to promote aeration. Incubate for <span class="time">1 hr</span> to permit expression of antibiotic resistance gene.</li> | |
| - | + | <li>Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate <span class="volume">200μL</span> but appropriate plating volume depends on efficiency of the transformation.</li> | |
| - | + | <li>Incubate plate overnight at <span class="temp">37°C</span>.</li> | |
| - | + | <li>Leave remaining SOC-cell mixture on the benchtop <span class="time">overnight</span>.</li> | |
| - | + | <li>If you don't have any transformants, plate the rest of the transformation in the morning.</li> | |
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 20:17, 22 September 2010
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
- 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.
Reference
adapted from http://openwetware.org/wiki/Knight:Electroporation
