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Team:UCL/Project/Protocols
From 2013.igem.org
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<p class="minor_title">Stable Transfection Of Adherent Cells</p> | <p class="minor_title">Stable Transfection Of Adherent Cells</p> | ||
<p class="body_text"> | <p class="body_text"> | ||
| - | This protocol is for the stable transfection of eukaryotic adherent cell types in a 6-well plate, with plasmid DNA that ought to be of as high a purity as possible. | + | This protocol is for the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate, with plasmid DNA that ought to be of as high a purity as possible. When transfecting multiple wells, make a 'master mix' with 10% of |
</p> | </p> | ||
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| - | <b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2). | + | <b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2)for 24 hours. |
</p> | </p> | ||
<p class="body_text"> | <p class="body_text"> | ||
| - | <b>3)</b> | + | <b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix. |
| + | </p> | ||
| + | <p class="body_text"> | ||
| + | <b>4)</b> Add 10µl | ||
</p> | </p> | ||
</div> | </div> | ||
Revision as of 11:45, 25 September 2013
Bacterial Lab Protocols
Stable Transfection Of Adherent Cells
This protocol is for the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate, with plasmid DNA that ought to be of as high a purity as possible. When transfecting multiple wells, make a 'master mix' with 10% of
1) The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.
2) Incubate cells in their normal growth conditions (37^0 C and 5% CO2)for 24 hours.
3) Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.
4) Add 10µl
Mammalian Lab Protocols
