Good day! I work with Cowpox virus (BAC). I did transfection (with Wild type). Used FuGene for that. After 24 h i could not detect any plaque formation. What is a problem may be?
Concentration of the BAC is around 5000 ng/µl
Protocol for transfection
2 µg DNA
4 µl FuGene
100 µl Opti Mem
Vortex 15 sec after adding Fugene. Leave for 15 min at RT. Add to cells.
I don't understand, are you just transfecting the viral DNA or is it competent virus? if competent (whole/infectious) you wouldn't need a transfection reagent so I'm assuming you just mean the viral DNA alone. first off I would dilute your BAC to around 0.5 mg/mL so you can pipette a larger volume and be more accurate. Also, have you tried different transfection reagent:DNA ratios? currently you're using a ratio of 2, try a ratio of 4 or 6 and observe for any plaques. if that fails, you may need to try a different transfection reagent. Search for a reagent that has been validated on the cell line you are trying to transfect.
I hope this helps you out.
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