I now need to co transfect two plasmids A and B, but I have been unable to transfer plasmid A and Western blot cannot detect the HA tag protein expressed in plasmid A. I can successfully transfer the A or B plasmid alone.
I used Thermo Fisher's reagent, Lipo 3000, to transfect according to the instructions: Solution A: Add 43ul Lipo3000 to 500ul Opti MEM and mix well; B solution: 14ug of plasmids A and B were added to 500ul of Opti MEM containing 56ul of P3000; Mix solution A and solution B well and incubate at room temperature for 15 minutes; Finally, add dropwise to the culture dish with 293 cells seeded on the first day; Change the liquid the next day; Collect protein samples in 3 days. My western blot still cannot detect the HA tag protein expressed by my plasmid A, but it can detect the target protein. My professor told me that this is because cells inherently express my target protein, and if the tag protein cannot be detected, it indicates that the plasmid has not been successfully transfected. So how should I solve this problem? Should we switch to a different cell line? Or is there a problem with my transfection process? thank you
Hello,
Actually, I don't know what is the ratio that recommended for 293 cells, I use 293ft cells which is very similar, and I use 2 uL of P3000 for each 1ug of DNA which is the same as you calculated but for lipofectamine, I use 3 uL of lipofectamine for each 1 ug of DNA, that should be 84 uL. Also, I leave the mixture for only 5 minutes (not 15) after mixing and I added directly to the cells as you described. other hints are to vortex solution A and use prewarmed optimum media, for soultion B, you mix only by gentle pipetting.
If in the same experiment you have one plasmid that is transfected and the other not I would suspect that something is wrong with the second plasmid; errors in the sequence? bad prep? wrong plasmid!! you can try cotransfection with a plasmid expressing a GFP for example to check for transfection.
As you already proved that transfecting either plasmid A or B works separately, but only failed with co-transfection, it is recommend to transfect one time each but in a time-dependent manner. For example, transfect plasmid A, wait for at least 1 h than apply DNA-lipo mixture B or vice-versa and test each condition for the best condition. I also agree with other says, check your plasmids DNA quality before any downstream application, when nicked DNA become more than supercoiled form, it substantially increase transfection difficulty.
Best.
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