Difference between revisions of "Team:TU Dresden/Notebook/Protocols/Electroporation"
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+ | <li style="margin-bottom: 10px;line-height:1.8;">Set up an electroporator at 1,350 V.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Add 1 μL of each plasmid to a different tube of electrocompetent cells.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Add the whole mix into a precooled electroporation cuvette, carefully knock the cuvette on the table to remove air bubbles and dry the metallic sides of the cuvette.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Place the cuvette into the holder of the electroporator, insert it and push 2 times the "pulse" button.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Take the cuvette out of the electroporator and add 1 mL of pre-heated LB medium without antibiotics into the cuvette, mix carefully by slowly pipetting up and down, and transfer the solution back into an Eppendorf tube.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Incubate the 37 °C (or at other temperatures depending on the sensitivity of the plasmid) for 45-60 minutes.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Streak out 50 μL of electroporated cells onto an LB plate containing the specific antibiotic of the plasmid.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Centrifuge the Eppendorf tube for 1 minute at 7,000 g and remove roughly 900 mL of the supernatant.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Resuspend the cell pellet in the rest of the supernatant and streak it on a new LB plate containing the specific antibiotic.</li> | ||
+ | <li style="margin-bottom: 10px;line-height:1.8;">Incubate overnight at the specific temperature of the <i>E. coli</i>.</li> | ||
+ | </ol> | ||
</html> | </html> |
Latest revision as of 14:30, 10 September 2015
Electroporation
- Set up an electroporator at 1,350 V.
- Add 1 μL of each plasmid to a different tube of electrocompetent cells.
- Add the whole mix into a precooled electroporation cuvette, carefully knock the cuvette on the table to remove air bubbles and dry the metallic sides of the cuvette.
- Place the cuvette into the holder of the electroporator, insert it and push 2 times the "pulse" button.
- Take the cuvette out of the electroporator and add 1 mL of pre-heated LB medium without antibiotics into the cuvette, mix carefully by slowly pipetting up and down, and transfer the solution back into an Eppendorf tube.
- Incubate the 37 °C (or at other temperatures depending on the sensitivity of the plasmid) for 45-60 minutes.
- Streak out 50 μL of electroporated cells onto an LB plate containing the specific antibiotic of the plasmid.
- Centrifuge the Eppendorf tube for 1 minute at 7,000 g and remove roughly 900 mL of the supernatant.
- Resuspend the cell pellet in the rest of the supernatant and streak it on a new LB plate containing the specific antibiotic.
- Incubate overnight at the specific temperature of the E. coli.