Infection with pMMP retrovirus

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Infection

  • Plate GM6914 cells onto 10-cm dishes.
  • Grow to ~50% confluency.
  • Thaw one aliquot of pMMP viral supernatant (SN) on ice

(may take several hours!)

  • Mix 50 ml DMEM/15% FCS with 1 ml 100X polybrene

(result: “2X polybrene medium”).

  • Only 5 ml of the 2X polybrene medium is needed per infection,

mix only as much as needed.

  • Add 5 ml 2X polybrene medium to 5 ml viral SN.
  • Incubate briefly at 37 degrees.
  • Rinse GM6914 cells once with PBS.
  • Aspirate PBS.
  • Add virus/polybrene to cells.
  • Incubate at 37 degrees/ 5% CO2 for 4-6 hours.
  • Change medium using DMEM/15% FCS.


100X Polybene stock sol’n

  • Weigh in 80 mg polybrene (Hexadmethrine bromide, Sigma H-9268).
  • Dissolve in 50 ml DMEM.
  • Filter through 0.2 um and store frozen (-20 degrees) in single-use aliquots (1 ml).

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