Rescuing from bacterial contamination? - (Nov/18/2014 )

I performed nucleofection on T47D and MDA-MB-231 cells with linearized HR and NHEJ plasmids. In my infinite wisdom, I decided not to put antibiotic in my medium (perhaps just to keep check on preventing Mycoplasma contamination). What I did not expect was that the transfected DNA (which had been purified with the QIAEX II and dissolved in water, not in TE) was carrying bacteria (consider this a dirty secret learned, because now I have to think about preventing Mycoplasma contamination in future experiiments in which I would put pen/strep in my medium).

When I checked the plate 18 hours after transfection, things looked good. A few hours later, though, I noticed some turbidity, although the phenol red had not changed color (parental lines were fine, meaning the problem not the medium). I immediately washed the wells of the 6-well plate five times with PBS, then added medium containing normal concentration of pen/strep (100 U/mL) and my selective G418, which should also have antibacterial activity, at 1 mg/mL. This seemed to cut down dramatically on the bacteria, and after replacing the medium again after 2 hr, the stragglers seem to be gone. The cells look intact under brightfield, but I'm going to wait until tomorrow. I really don't want to do another nucleofection, because I am out of electroporation cuvettes. Is it possible that I caught it in time and saved my cells? Also, is it possible that the LPS released from the bacteria may have affected my constructs to the point that the I-sceI sites may have been compromised and would allow for reexpression of GFP?  I have planned on sequencing my insert in the event of any integration.  Thanks for any response.

First off - pen/strep won't prevent mycoplasma infection - they don't interfere with the right pathways to affect mycoplasma species... adding them only works for bacteria with cell walls.

It is very unlikely that you have got rid of the bacterial contamination by washing, but the G418 and pen/strep should work (so long as they aren't resistant... they could take up your plasmid though). The bacteria are unlikely to interfere with the DNA itself, but they will induce interferon production, which should (IIRC) in turn induce anti-infection responses such as an increase in DNA damage response. The only way to be sure is to examine those pathways and see.

I would still advocate that you throw these ones out and make more if at all possible (perhaps keep these as a backup), pen-strep often won't fully get rid of infections, but rather just suppresses growth such that there is a hidden contamination which can affect future results.

To get rid of bacteria in plasmids you can add a drop of chloroform and shake vigorously. then leave the mix to separate. The chloroform attacks cell membranes thereby killing the bacteria.

Thanks for the response.  Actually, it did end up being the medium, because when I came in today, I saw that the parental lines were infected (even though they didn't show any signs of infection yesterday, compared to the transfected cells.  Weird).  That being said, the transfected cells incubating in medium containing pen/strep and G418 did not show any signs of infection.  I am going to wash the cells in new medium containing pen/strep and incubate with it and G418 selection just to get it into what should be uncontaminated medium.  And come to think of it, integration of the linearized plasmid into the genome does require some form of DNA damage and repair anyways.  Whether the acute interferon signaling (which hopefully should be reduced to basal expression with the killing of bacteria and removal of LPS) will induce long-lasting effects is something I need to see, and of course I can check for issues with the plasmid visually to see GFP re-expression (which shouldn't be there without I-sceI or maybe even IR) and with sequencing to make sure everything is 100%.  Of course, I also bought new cuvettes, so hopefully I can get another transfection done this week before going to Florida for Thanksgiving.  The only question now is how long I can store linearized DNA in a frost-free freezer before it goes bad.

In theory, you can store it indefinitely. However, I always find it better to make fresh digests.