| |||||||
-Set up 60 mm dishes of P11 cells to be 100% confluent at time of infection.
-Remove medium from dishes, add 0.2 to 0.5 ml virus and adsorb for 30 – 60 minutes at room temperature, rocking dishes occasionally. For titration, test dilutions of 10-5 to 10-11.
-Add 10 ml complete DMEM-agarose overlay per plate and let cool. Add 1 ml normal P11 growth media to top so as not to allow agarose to dry out. Incubate at 37°
-Plaques should be visible within 4 – 5 days and should be counted for titration at day 7 and day 10.
To make complete DMEM-agarose:
-Prepare 2X DMEM.
-As needed prepare 2X DMEM plus additives: to 82 ml 2X DMEM add 10 ml Horse serum, 2 ml L-glutamine, 2 ml pen-strep, 2 ml fungizone and 2 ml autoclaved 5% yeast extract and sterile filter
-Autoclave 1 g agarose in 100 ml water. Microwave prior to use and keep at 45 – 50° water bath until use.
-Prior to adding to plates make a 1:1 mixture of the agarose and complete DMEM