Infection with pMMP retrovirus



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Infection

 * Plate GM6914 cells onto 10-cm dishes.


 * Grow to ~50% confluency.

(may take several hours!)
 * Thaw one aliquot of pMMP viral supernatant (SN) on ice

(result: “2X polybrene medium”).
 * Mix 50 ml DMEM/15% FCS with 1 ml 100X polybrene

mix only as much as needed.
 * Only 5 ml of the 2X polybrene medium is needed per infection,


 * 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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