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Team:BIOTEC Dresden/Protocols:Electroporation
From 2010.igem.org
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{{Biotec_Dresden/Header}} | {{Biotec_Dresden/Header}} | ||
<html> | <html> | ||
| - | < | + | <body> |
| - | + | <h2>Materials</h2> | |
| - | </ | + | <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> | ||
| + | <h2>Procedure</h2> | ||
| + | <ul> | ||
| + | <li>Chill electroporation cuvettes, DNA samples and tubes on ice.</li> | ||
| + | <li>Place LB-agar plates in 37°C incubator twarm.<li> | ||
| + | <li>Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.</li> | ||
| + | <li>If electrocompetent cells are not already in individual aliquots, then aliquot out intpre-chilled 0.6mL tubes.</li> | ||
| + | <li>Turn on electroporator and set voltage teither 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).</li> | ||
| + | <li>Dial a P2 pipetman teither 1 or 2µL depending on the salt content of your DNA sample and . Use 2µL for samples that have been purified in some way.</li> | ||
| + | <li>Dial a P200 pipetman t50µL or whatever volume of electrocompetent cells you want tuse. Usually 20-50µL.</li> | ||
| + | <li>Dial a P1000 pipetman t950µL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.</li> | ||
| + | <li>Pipet 1-2µL of DNA sample and add telectrocompetent cells. Swirl tip around gently in cells tmix DNA and cells. Dnot pipet up and down. </li> | ||
| + | <li>Place cells back on ice tensure they remain cold.</li> | ||
| + | <li>Transfer cell-DNA mixture tcuvettes using P200 pipetman. Try not thandle cuvette base tomuch sthat it stays cold.</li> | ||
| + | <li>Tap the cuvette on the counter gently sthat cells are at the bottom and tremove 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 sthat 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 tprevent cells from dying off.</li> | ||
| + | <li>Transfer SOC-cell mixture tchilled eppendorf tube.</li> | ||
| + | <li>Chill sample on ice for 2 mins tpermit the cells trecover.</li> | ||
| + | <li>Transfer eppendorf tube t37°C incubator and shake tpromote aeration. Incubate for 1 hr tpermit expression of antibiotic resistance gene. </li> | ||
| + | <li>Plate transformation ontprewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200µL but appropriate plating volume depends on efficiency of the transformation.</li> | ||
| + | <li> Incubate plate overnight at 37°C.</li> | ||
| + | <li> Leave remaining SOC-cell mixture on the benchtop overnight.</li> | ||
| + | <li> If you don't have any transformants, plate the rest of the transformation in the morning.</li> | ||
| + | </ul> | ||
| + | </body> | ||
</html> | </html> | ||
[[Category:BIOTEC Dresden/Protocol]] | [[Category:BIOTEC Dresden/Protocol]] | ||
{{Biotec_Dresden/Bottom}} | {{Biotec_Dresden/Bottom}} | ||
Revision as of 16:36, 13 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 twarm.
- 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 intpre-chilled 0.6mL tubes.
- Turn on electroporator and set voltage teither 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).
- Dial a P2 pipetman teither 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 t50µL or whatever volume of electrocompetent cells you want tuse. Usually 20-50µL.
- Dial a P1000 pipetman t950µL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.
- Pipet 1-2µL of DNA sample and add telectrocompetent cells. Swirl tip around gently in cells tmix DNA and cells. Dnot pipet up and down.
- Place cells back on ice tensure they remain cold.
- Transfer cell-DNA mixture tcuvettes using P200 pipetman. Try not thandle cuvette base tomuch sthat it stays cold.
- Tap the cuvette on the counter gently sthat cells are at the bottom and tremove any air bubbles.
- Wipe off excess moisture from outside of cuvette.
- Place in chamber of electroporator.
- Slide the chamber in sthat 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 tprevent cells from dying off.
- Transfer SOC-cell mixture tchilled eppendorf tube.
- Chill sample on ice for 2 mins tpermit the cells trecover.
- Transfer eppendorf tube t37°C incubator and shake tpromote aeration. Incubate for 1 hr tpermit expression of antibiotic resistance gene.
- Plate transformation ontprewarmed 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.
