No Retrovirus using the Phoenix - Ampho cells - (Oct/28/2008 )

Hi, I've been trying to produce retroviruses using the PHoenix-ampho cells. I am transfecting the phoenix cells with 10 ug of my viral vector using the CaPO4 method. I collect the supernatant 48 hours after transfection. I then applied this viral supernatant to fibroblasts (to test viral infection) and I got nothing. I thought it would be an easy method to produce retrovirus. Are there any suggestions? I even tried using chloroquine.

Thanks!

Do you have a any sort of control viral construct you could transfect in to test for transfection efficiency.. Once you know the DNA is getting in fine (10ug seems like a LOT, but I haven't read the phoenix protocol properly) then you know it just isn't producing a virus, or not infecting.
polybrene can assist with infections. - if it doesn't work after that then it may just be a dud construct - or toxic to the cells.