Infection with pMMP retrovirus: Difference between revisions
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=='''Infection'''== | =='''Infection'''== | ||
Plate GM6914 cells onto 10-cm dishes. | *Plate GM6914 cells onto 10-cm dishes. | ||
Grow to ~50% confluency. | *Grow to ~50% confluency. | ||
Thaw one aliquot of pMMP viral supernatant (SN) on ice | *Thaw one aliquot of pMMP viral supernatant (SN) on ice | ||
(may take several hours!) | (may take several hours!) | ||
Mix 50 ml DMEM/15% FCS with 1 ml 100X polybrene | *Mix 50 ml DMEM/15% FCS with 1 ml 100X polybrene | ||
(result: “2X polybrene medium”). | (result: “2X polybrene medium”). | ||
Only 5 ml of the 2X polybrene medium is needed per infection, | *Only 5 ml of the 2X polybrene medium is needed per infection, | ||
mix only as much as needed. | mix only as much as needed. | ||
Add 5 ml 2X polybrene medium to 5 ml viral SN. | *Add 5 ml 2X polybrene medium to 5 ml viral SN. | ||
Incubate briefly at 37 degrees. | *Incubate briefly at 37 degrees. | ||
Rinse GM6914 cells once with PBS. | *Rinse GM6914 cells once with PBS. | ||
Aspirate PBS. | *Aspirate PBS. | ||
Add virus/polybrene to cells. | *Add virus/polybrene to cells. | ||
Incubate at 37 degrees/ 5% CO2 for 4-6 hours. | *Incubate at 37 degrees/ 5% CO2 for 4-6 hours. | ||
Change medium using DMEM/15% FCS. | *Change medium using DMEM/15% FCS. | ||
'''100X Polybene stock sol’n''' | '''100X Polybene stock sol’n''' | ||
Weigh in 80 mg polybrene (Hexadmethrine bromide, Sigma H-9268). | *Weigh in 80 mg polybrene (Hexadmethrine bromide, Sigma H-9268). | ||
Dissolve in 50 ml DMEM. | *Dissolve in 50 ml DMEM. | ||
Filter through 0.2 um and store frozen (-20 degrees) in single-use aliquots (1 ml). | *Filter through 0.2 um and store frozen (-20 degrees) in single-use aliquots (1 ml). | ||
[[Kupfer Lab:Back Door|back to protocols]] | [[Kupfer Lab:Back Door|back to protocols]] | ||
Revision as of 10:36, 15 August 2006
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).
