After transfection and passaging I loose my virus - (Jun/24/2008 )

Hello,

I need help for this problem if any of you has a suggestion please let me know. I'm trying to reconstitute my virus BAC after transfection Calcium phosphate. I got plaques but after 2-3 passages of my cells the virus disappear and no more plauqes are visible. Do you have any idea?

Thanks in advance for your answer

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What virus are you using?
How long after transfection can you see plaques forming??

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It's an Herpesvirus and I should see plaques normally after 6-7 days but sometimes they are not clearly visible and I need to blindly pass my cells before seeing the plaques on fresh cells. But then after passaging 1 or 2 times the virus is gone

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Which herpesvirus??

When you passage onto fresh cells, do you increase the cell number?? ie are you passaging from, say a 6 well to a flask? or are you passaging onto approx same number of cells??

Also, could you just replenish the media, and not passage your cells until the virus has infected a greater number of your cells??

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After transfection of a 6wells I leave the cells for 5-7 days before passaging them on New T75 so yes I increase the number of cells. The virus is MDV marek's disease virus
I have to pass my cells after 5-7 days otherwise they don't look happy and start to die.
Last week I could see big plaques on my T75 and then I passed them on fresh cells (1T75 of infected cells was mixed to 1T75 of fresh cells and 3T75 were prepared from that but till now no visible plaques can be seen)

Thanks

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