Submitted by Lauren French (download pdf)

Poly-dL-Ornithine (PORN) Solution (Sigma-Aldrich Product # P-8638) preparation and coating of plates:

 

  • Remove PORN from freezer and allow it to come to room temperature.
  • Add 1.668g Boric Acid (Sigma-Aldrich Product # B-0394) to 180mL triple distilled water and stir to dissolve.
  • Adjust the pH to 8.3 using NaOH.
  • Add 90mg room temperature PORN and stir to dissolve.
  • Sterile filter this solution in the hood.
  • Make sure 96-well plates (Falcon Product #35-1172) and 35mm dishes (Falcon Product # 35-10080 are still sealed in original sterile packaging, and do not open until they are in the hood.
  • Add 100µL per well on 1.5mL per dish of the above PORN solution.
  • Wrap tightly in parafilm and store overnight at 4ºC.
  • The next day, rinse the plates or dishes three times with sterile filter water, using 200µL or well or 1 mL per dish for each wash.  Be careful not to let the dishes/wells dry out in between rinses.
  • After the third rinsing, shake each dish or plate against the side of the waste bucket to remove as much water as possible.
  • Then slam the dish or plate onto a few stacked kimwipes to remove even more water.
  • Allow them to dry completely in the hood, then store at 4ºC until ready to use.  The plates and dishes do not need to be wrapped in parafilm during storage, but make sure they do not open, to prevent contamination.
  • The PORN solution listed above will make enough to coat approximately 9, 96-well plates or 120, 35 mm dishes.  It can easily be adjusted to coat more or less plates or dishes.

 

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