We use mammalian expression plasmids that show repeated (but inconsistently) problems as failure to transfect (depending on transfection reagent, FuGENE or PEI), uneven transfection pattern on WB, gelly consistence after several months, but even as one month old. Common solution is just to make new ones (or try to boil them for 10 minutes, but allegedly that doesn't help that much), but the problems keep happening once in several months to different people, I suspect there to be a systemic problem.
I would normally start at the beginning and try to check everything, but due to certain resons I'm not allowed to. Also, there are several things done not in plasmid-GLP way, some of which I tryied to avoid, but others I can't.
Plasmids are isolated from E.coli. on a Invitrogen HiPure column, but it is overloaded (recommended volume for high copy plasmid is 15-25 ml of culture and we use 100 ml). Nonetheless the yields are good and 260/280 purity as measured on Nanodrop is 1.9 (the 260/230 is also fine). And they seem to work, at least at the start, with storage concentration around 1 ug in low EDTA TE. Commonly they are disolved overnight at fridge and measured, diluted and stored in -20, thawed for each use (which could be like once a week). But mostly they would be overdried before that, as this is difficult to prevent with processing several plasmids at once.
What I did try was to carefully get rid of any ethanol wash remains, try not to overdry pelet and keep it constantly in 4 C (and before that 1 hour on 37 C), and so far they seem to work (with PEI). But also I needed to see if that has any effect.
I have limited means to test them, I had only a few REs to use, I can run uncut on gel, sequence with common plasmid primers and test different conditions of storage, but that's all. So I mainly focus on bacterial gDNA contamination, plasmid size and plasmid conformations.
My first (frozen-unfrozen, a bit gelly after a while) batch I run on gel and seen a huge bright wide blob in between where the plasmids and gDNA should be and visible signs of undissolved DNA (the bands with these rectengular-like tail edges). That and something about frequent freezing-thawning cycles possibly creating a small microenviroments around DNA, that could affect solubility, I decided to keep them in fridge further on, as they should be stable anyway for such short time we use them.
The I used a single cutter to check the size and they have the expected 8 kb, also fully cut they do not exhibit any smear or blob, so I concluded that the smear had to be plasmids (possibly some multimers??).
Anyway I never seen the blob since, while I keep them in fridge, so that should have been gone.
But now the very stupid question at the current end of my testing, when I run 100 ng on 0.8% agarose (0.5x TBE buffer) I see the most bright conformation to be much higher that 8 kb (and 5,5 kb for empty) even though the supercoiled should run even lower than that. My colleague told me, that that's the effect of EtBr binding and it is the supercoiled, but the same happens when I decrease the amount of EtBr to 1/2 and I'm still not sure, that it could cause such a shift and I didn not ever seen that before on plasmid checks.
I was wandering then if that means that the most abundant now is not the supercoiled variant. Or maybe not using TAE for plasmids (that I can change).
I'm aware that transformation efficiency varies with different conformation and used method, usually supercoiled are preferable. Any ideas welcome.
(lines 1-2, 8 kb plasmid, 3-4 something around that size too, 5 is 5.5kb empty backbone)
