PEI Stocks

Materials

  • Linear PEI (located in “Chemicals” tray in Oaken 4ºC)

  • Hydrochloric acid (HCl), to balance pH

  • Elga water

  • 0.22 µm filter (recommended 500 mL size)

Warning

Ensure you use the linear PEI, not the branched version!

Dissolve and Aliquot PEI

  1. Before beginning, rinse all glassware and stir bar with Elga water.

  2. In a beaker on a stir plate, combine 1 mg PEI powder per 1 mL Elga water.

  3. While stirring, slowly add HCl to dissolve the PEI and achieve a final pH of 7.0.

    • PEI will not dissolve well in a basic solution, so slowly add acid to facilitate this process.

    • Add acid a few drops at a time to not overshoot. Total acid required is ~1 mL of 2.5M HCl.

    • After each addition, wait several minutes for the solution to stabilize. After pH 7.0 is reached, wait an extra 10 min to ensure the pH has not drifted.

  4. In a BSC, filter sterilize the PEI solution through a 0.22 µm filter (the 500 mL ones are appropriate).

  5. Make 1 mL aliquots of PEI in sterile 1.7 mL Eppendorf tubes.

  6. Store the aliquots in Nokk (-80ºC, 2nd shelf from the top). Determine the optimal PEI ratio before use.

Test PEI Ratio

The optimal mass ratio of PEI to DNA for transfection can vary across batches. While more PEI typically improves transfection efficiency, it also increases toxicity. We perform a test transfection using varying amounts of PEI to determine the optimal μg PEI:μg DNA ratio. Previous tests have used ratios ranging from 1:1 to 6:1. Previous optimal ratios have been 4:1 or 5:1. The following protocol tests transfection efficiency in 6-well plates for ease of pipetting PEI volumes. Smaller well plates and volumes may be used if the number of cells and the amount of DNA are decreased in proportion to the decrease in culture surface area.

  1. The day before transfection, seed 1 million 293T cells in 1 well of a gelatin-coated 6-well plate for each ratio to be tested.

  2. Prepare a midiprep of a plasmid tht expresses a single fluorescent protein under the control of a strong constitutive promoter such as CMV or EF1α. Plasmids such as pKG1923 (pSHIP-EFS-mGreenLantern-bGH) or pKG2340 (pSHIP-CMV-mRuby2-bGH) are appropriate would work, along with any other simple plasmid. You will need 2 μg of DNA per ratio tested.

  3. The day of transfection, prepare a knockout DMEM + PEI mix for each ratio to be tested. Add PEI to KO DMEM, invert to mix, and wait 10 minutes.

    • 1:1 - 200 μL KO DMEM + 2 μL PEI (1 μg / μL)

    • 2:1 - 200 μL KO DMEM + 4 μL PEI (1 μg / μL)

    • 3:1 - 200 μL KO DMEM + 6 μL PEI (1 μg / μL)

    • 4:1 - 200 μL KO DMEM + 8 μL PEI (1 μg / μL)

    • 5:1 - 200 μL KO DMEM + 10 μL PEI (1 μg / μL)

    • 6:1 - 200 μL KO DMEM + 12 μL PEI (1 μg / μL)

  4. After 10 minutes, add 2 μg of plasmid DNA to each KO DMEM/PEI Mix. Invert to mix and wait 10 minutes.

  5. After 10 minutes, add the entire KO DMEM/PEI/DNA mix dropwise to a single well of the 6-well plate. Repeat for all tested titrations.

  6. Return cells to the incubator.

  7. The next morning, replace the media with fresh DMEM + 10% FBS.

  8. 2 days after transfection, assess transfection efficiency using the Keyence or the Attune.

Reference

https://www.addgene.org/protocols/transfection/