Hi everyone
I'm trying to titre some stocks of adenovirus that I isolated from infected 293a and I need a protocol to see the infection efficiency as the plaque forming assay
However, I tried some protocols using 293a cells, but it did not give consistent results.
problem: Too many cells, no plaque (cells grow over the plaques), not many cells.
For example:I seed 293a cells (0.1 M/well in 6 well plate) and infect on the second day,, I incubate for 6 days ,,, then I don't see any plaques,,, as cells are over grew or no infection ,,,
Please any expert can help me with this ,,,
Many thanks
Hi,
I have attached a protocol.
Please let me know if you have better results
Thanks
Angel
Hi
Thank u Angel
I tried it but no plaques can be seen
As the E1 gene is deleted from the virus
So do you have any alternative way ?
Thanks
Do you have any reporter gene expressed by the vector, like GFP or beta-gal? Without fluorescent or other labelling plaques can be difficult to relably recognize, which can lead to inconsistent results in repeated experiments.
Dilution factor also matters, if you count the wells with higher dilutions, where you have only a few plaques, the error will be high. Go for lower dilutions, i.e. for higher number of expected plaques so your error will be lower.
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