I am trying to co-transfect 3 plasmids into macrophage cell line (RAW264.7). I can transfect single plasmid successfully and can detect with immunoblotting. But it is very hard for me to do 3-plasmid co-transfection.
Does anybody have good idea or advice? Thank you so much.
Dear Xianfeng, are you using the same total amount of plasmid? How are you proceeding in detail?
Hi Wolfgang, thanks for your input.
I am using SuperFect to transfect. Based on the standard protocol, 2ug DNA is good for 6-well plate, so I added total 2ug plasmid DNA, 0.6ug for each. All procedures follow the standard protocol.
If the ratio of plasmid to transfection agent is ok, then actually noting should go wrong (usually, more than one copy of the plasmid gets into a cell anyway, so a mix should not make trouble). Can you add some GFP plasmid to check the actual transfection in order to make sure the problem is not occurring in a later step (i.e. detection).
To make sure the cells themselves have no problems (virus, mycoplasma etc) transfect single plasmids (include GFP for convenience) at the same time, maybe also to other cells known to make no problems (eg 293HEK, 3T3 and so on).
Just in case one of the plasmids is toxic or incompatible: Try combinations of 2 plasmids.
I think the plasmids and cells are good, because I did single transfection simultaneously. Also I just went to check and found all plasmids have the same promoter, CMV promoter, to drive the target genes.
You say "incompatibale", you mean promoter or something else? Thank you so much.
Hi Mark, I did not try electroporation yet. But I am sure each plasmid expresses well when transfected alone.
When you look at the transcribed sequences, are there reverse complements originating from different plasmids? They might hybridize and lead to transcript destruction.
Thank you, Wolfgang. I plan to transfect with different combinations.
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