Method for detecting rAAV - (Apr/11/2007 )
I detect the transgene in the packaging cell lysate by PCR 48 hours after transfection. The lysate was treated with DNase1 and divided into 2 tubes. One was subjected to proteinase K digestion, and the other left untreated. Only the proteinase K treated sample can PCR amplify the transgene. Can I speculate that the transgene was successfully packaged in the virus?
yes, the transgene should be packaged.
Thank you Scolix.
I was uncertain whether the transfected plasmids were associated with histone or other proteins. So Proteinase K treatment may be necessary for PCRing any DNA sequence in crude cell lysate. In my experience, this was true for PCR amplifying genomic sequences. Even without DNase1 treatment, I could't detect the transgene by PCR in the crude cell lysate of transient transfected cells.