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Q I have a plasmid in TE-buffer, but I'm not sure that it's sterile anymore. Is there a quick and easy way to sterilize my DNA? Can I re-precipitate, heat, or filter it? If so, what kind of filter can I use so that I won't lose too much DNA?
A That depends on what type of contamination you have and what downstream applications you have planned for the DNA. If you have nuclease, protein, or bacterial contamination, you can heat the plasmid solution to 80°C for 20 minutes. If the contamination is from another DNA, gel purification may work. But remember that plasmid DNA is supercoiled and therefore looks like multiple bands of different sizes in the gel. It will be hard to know if you are purifying the plasmid DNA or the contamination.
Q The DNA is intended for transfection and I think it might have bacterial contamination because I accidentally opened my vial outside the hood. Do you know how heating will affect the plasmid depending on size?
A1 You could just re-transform it into bacteria and extract the sterile DNA.
A2 Why bother with another transformation and plasmid prep if you have the DNA already? Heating the DNA is fast and easy.
A3 You could ethanol precipitate it, rinse it with 70% ethanol, and redissolve it in endotoxin-free TE. The 70% ethanol should sterilize the DNA.
Q I'm afraid that I will lose too much material. So from the options of heating and precipitation, which would you choose to get rid of bacterial contamination without losing the DNA?
A The traditional way to sterilize DNA is by precipitation. If done properly (be sure not to aspirate the pellet), you should not have any loss as long as you have greater than 1 ng/µl in your starting material. Some organisms that survive heating will be destroyed by alcohol. In practice, I don't worry very much about sterility for transfections that will be lysed after 48 hours and wouldn't bother to sterilize otherwise clean DNA that had been opened outside the hood. I'm not saying this is necessarily the best practice, but we haven't had problems. (Of course, you should still be careful for the sake of colleagues who are carrying on long-term cultures in the same incubators.)
Q I'm going to use this for a longer culture, so I understand that it needs to be sterile. So if I need to precipitate again, will it work with only ethanol or do I need to add salt as well to get the DNA to precipitate easier?
A Add 1/10 volume of 3 M sodium acetate and 2.5 volumes of ethanol. After spinning, rinse the pellet with 70% ethanol. Air dry and re-dissolve the pellet in endotoxin-free TE.
If you are preparing the DNA for stable transfection, you need to linearize the plasmid. Purify the linearized plasmid with a spin column and elute with endotoxin-free TE. You need not worry about sterilizing the plasmid DNA before restriction digestion because the restriction enzyme and the buffer that comes with it may not be sterile anyway.
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