Dear all,
I am trying to isolate recombinant Modified Vaccinia Ankara (MVA) clones from plaques in the presence of an agarose overlay.
I transfected the MVA-infected DF-1 cells with a shuttle vector containing the GFP-encoding genes, then harvested and lysed the transfected cells and used the lysate to infect DF-1 cells in a 6-well plate. After culture, quite a few cells expressing green fluorescence can be observed, suggesting the recombinant virus was produced.
In order to isolate the recombinant virus carrying the GFP gene, 2 h after the lysate of the transfected cells mentioned above was added, the medium was removed, and an agarose overlay, made by mixing 2% LMP agarose (Invitrogen 16520-100) and 2x DMED, was added to the wells. After the agarose was solidated at room temperature, the cells were cultured, but only very few fluorescent cells can be observed.
Obviously, the presence of the agarose overlay affected the replication of the recombinant virus with GFP, making it difficult to form virus plaques for isolation.
Do you have any suggestions to solve the problem? I will deeply appreciate it if you could help.
Best regards and thank you!
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