flashBAC Manual - Oxford Expression Technologies

the virus titre has not been checked, there may bedisappointment when gene expression is very low orundetectable.• The most common cause of failure to detect foreigngene expression is using a stock of virus in which thetitre is assumed to be high but, when titrated byplaque-assay, turns out to be very low!• For most purposes a titre of 5 x 10 7 pfu/ml or higher isadequate. A titre of less than 10 7 pfu/ml will notnormally be sufficient for expression studies.• Virus may also be amplified in monolayer cultures,with the cells seeded in T75 or T150 flasks to formsub-confluent monolayers. The medium is removedand the cells infected with virus (use 100 –200 µl ofthe seed stock virus from the co-transfection dilutedto 0.5 – 1.0 ml with medium). Allow the virus toadsorb for 1 hour and then remove. Replace withfresh medium (10-15 ml for a T75 and 30 ml for aT150). Allow the virus to replicate until the cells arewell infected – 3-5 days. Harvest the medium as thesource of amplified virus inoculum and store/titrate asdescribed above. The titre of virus amplified in thisway is not usually as high as virus amplified in shakeculture.OXFORD EXPRESSION TECHNOLOGIES 30