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>
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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  
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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 all solutions. Include negative, and where possible, a positive control.
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<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.
<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.
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<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.
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<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.
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<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml.
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<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well.   
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<b>8)</b> Change medium and wash with PBS. 
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<b>9)</b> Incubate for 24-48 hours.
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<b>4)</b> Add 10µl
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<b>9)</b> Assay fro gene expression.
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Revision as of 11:56, 25 September 2013

There are two solutions to \(ax^2 + bx + c = 0\) and they are $$x = {-b \pm \sqrt{b^2-4ac} \over 2a}.$$

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 all solutions. Include negative, and where possible, a positive control.

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 of superfect (SF) reagent to the solution. Vortex for 10 seconds.

5) Incubate at room temperature for 5-10mins to allow for complex formation.

6) Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml.

7) Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well.

8) Change medium and wash with PBS.

9) Incubate for 24-48 hours.

9) Assay fro gene expression.

Mammalian Lab Protocols

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