Hi everyone.
I transfected sf9 insect cells by my purified DNA bacmid. After 3 days post-transfection, I could not be able to see any cells infection signs; so I collected my P1 virus stock expecting that cells have been infected by virus. Then, now I want to check whether I got virus containing my Gene Of Interest on it.
So, can I run PCR using the specific primers targeting my GOI? Or, how can I check it?
this link is useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689143/
egards
Hello everyone
thank you for yours recommendations.
I run a normal PCR, but I didn't get any band. As you suggested me, I will run a RT-qPCR.
Anyway, I want also to notice that my protein interest is a non-secreted protein, it is an intracellular protein. Is there any specificity in the transfection protocol?
Thank you?
For doing the PCR, it is recommended to use M13Forward OR Reverse and Gene of interest Forward OR Reverse.
M13 primers are short and there is a possibility of non-specific annealling.
By doing this, you will ensure that you have a bacmid with insertion of your gene of interest.
Three days post-infection, you can harvest your cells and do the immunoblot. If you got the band, fine! If you got the faint band, then you can infect the cells for longer time, I would say 5 days. If you got no band you should work on the transfection as transfecting these large bacmids are not that easy. PEI-based transfection worked very well for me.
Hope this help.
I think you are supposed to be PCR to confirm whether your gene is into bacmid DNA prior the transfection. I strongly recommend Cellfectin reagent to perform transfection at least 5 days. Finally, you can check your protein on the membrane by western blotting which is very sensitive experiment. Good luck
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