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Protocol

Titration of Neo-r Retrovirus
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Titration of Neo-r Retrovirus Day -1 In 6-well panels, plate 5xlo4 NIH3T3 cells. In DMEM supplemented with 10% FCS. Plate one well per dilution of viral stock and controls. Depending on your estimate of the titer, make the dilutions accordingly, using 1 well per infection media, 10-1, 10-2 10-6 + control. Incubate cells 370 overnight. Day 0 Prepare 4 mg/ml polybrene solution in DMEM/FCS, this will be 1000x stock. Label eppendorf tubes with each dilution. Prepare dilution solution (DMEM/FCS) with polybrene at 4 ug/ml. Calculate the amount needed according to the number of dilutions you have. You will need 1 mg of infection media per well. Thaw viral-samples in 370 bath. Dilute viral stock in diluent using a 1/10 serial dilution scheme. Mix by pipetting, do not vortex. Remove media from NIH3T3 cells plated yesterday. Add 0. 5 ml of the infection media to the wells labeled with the corresponding dilution. Incubate 2 hours 370. Periodically tip the plates back and forther to spread the infection media over the monolayer. Remove the infection media. Add 2-5 ml DMEM/FCS. Incubate 20-24 hrs.
Day 1 Change to selection media; G418 at appropriate concentration (usually around 500 ug/ml - 1 mg/ml active) . A kill curve should be constructed to determine the appropriate concentration of G418. Incubate 8-14 days until colonies are visable under the microscope. Change the media every 3 days. Days 8-14 Stain to visualize colonies; Aspirate media and add 0.5 ml methylene blue in MEOH (0.1 g/60 ml, filter through Whatman). Stir at room temp. for about 10 minutes with gentle swirling periodically. Rinse x5 stain art with H20- Air dry plates. Calculate titer/ml colonies x dilution x volume plated.
References: Cone, R.D., Grodzicker, T., Jaramillo, M. (1988) A retrovirus expressing the 12S adenoviral ElA gene produce can immortalize epithelial cells from a broad range of rat tissues. MCB . "Mann, R., Mulligan, R., Baltimore, D. (1983) Construction of a retrovirus packaging mutant and its use to produce helper free defective retrovirus. Cell 33: 153-159.

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