Hi, I have a set of plasmids ( midi-preps isolated using a standard kit). When I had freshly isolated them, I had resuspended the DNA pellet in TE buffer and I have been using these plasmids for some time now with satisfactory results. About 2 months ago I had to measure the concentration of one of them and it seemed higher than before, for some reason. (The 260/280 ratios seem fine). Since then I re-measured some of the ones I most often use and surprisingly the nano-drop shows me a higher reading for all of them. For eg: Previously if a plasmid had a conc of 1microgm/microlit now it shows me 1,5microgm/microlit. I ran the samples on an agarose gel and none of them show a smear ( which would be the case if the plasmid was degraded). Does anyone have an idea of why this would happen ? is it normal ? how can i avoid it. I usually store my working stocks at 4deg. in eppendorf tubes. Before every use I usually give it a short mix and then spin-down.
Plasmid at high concentration like 1mg/ml or higher is not fully dissolved. Some plasmids aggregate to form small particles. Over a long time, the complex dissolved to individual molecules, and thus increase the concentration.
If the plasmid is stored at -20C, there will be evaporation which also increase the concentration. It should be stored at -80C.
Plasmid at high concentration like 1mg/ml or higher is not fully dissolved. Some plasmids aggregate to form small particles. Over a long time, the complex dissolved to individual molecules, and thus increase the concentration.
If the plasmid is stored at -20C, there will be evaporation which also increase the concentration. It should be stored at -80C.
Most probably your plasmids might have formed soluble aggregates upon storage at 4 degree for long time. The concentration of plasmids or proteins is usually overestimated in the presence of soluble aggregates. You need to maintain your plasmids at -80 degree as suggested above by Chuandong Fan.
Alternatively, you can transform your plasmids into cloning host bacterial strains. Glycerol stocks of the strains carrying your plasmids can be maintained at -80 degree. Whenever you need plasmids, you can culture the bacteria, and isolate and purify plasmids.
Honestly, if they are working well I would't worry about it. The concentration could actually be higher due to evaporation of the TE buffer. Or the nano drop wasn't properly zeroed for one or both readings. Basically, if you are able to continue with your experiment I would just keep using them and not bother with any sort of trouble shooting. You should store your purified plasmid stocks at -20 or -80 and only keep them at 4 for short term (a month or less).
Did the volume change as well? If you store your plasmids at 4 degrees, then the TE buffer is likely to evaporate and this will give you a higher DNA concentration. I would store them at -20.
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