I transfect my RPE1's in an ibidi 8 well dish (small dish, holds 300 uL per well) - this involves seeding them the day before, and then transfecting them when they are at 80% confluency. The transfection process involves mixing the reagent with serum free media + DNA, incubating it in the microcentrifuge tube for 20 minutes, then replacing the cells media with this mix and supplementing it with new media (DMEM-F12 supplemented with 10% FBS and 1/5000th hygromycin B). To avoid cell death, we replace the media about 2 hours after transfection.
I've been doing this for a while but for the last month my lab mate and I have been experiencing bacterial contamination almost every single time we transfect (we can tell by the biofilm formation and intense yellow colour of media). There is never contamination after just seeding them into the plates (so the plates aren't the issue) but after transfection and replacement of media, the contamination shows up about 4 hours later.. so it's something at this part.
We have tried testing our transfection reagent, DNA samples, and SF-media by plating them with supplemented media (without cells) in a 24 well plate and keeping it in the incubator but no bacteria ever grew.
Is there a chance that our DNA could be contaminated but just doesn't grow bacteria without the presence of cells, or could something else be going on entirely? We are starting to feel very hopeless and any advice would be greatly appreciated! We're just not sure what else to try or where to go from here :(
Hi Nola,
Is there a chance the contamination is occurring during the centrifugation step?
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