I need to use MES buffer in my experiment, and I want them to be DNase free, can I UV sterilize the buffer without changing their properties?
UV sterilization is probably not a good idea because it could cause photodamage to the MES molecule. Moreover, it will not necessarily destroy DNase . You can filter sterilize the buffer with a 0.45 or 0.2 micron filter. To eliminate DNase activity, all you have to do is put in 1 mM EDTA and don't add any divalent metal ions, because DNase is activated by Mg2+.
Adam B Shapiro Hi Adam, thank you for telling me these, but I'm about to use the MES buffer in a enzyme reaction, so I'm afraid that adding EDTA might affect my enzyme activity.I thought the low Ultraviolet absorption of MES buffer would allow them to stay stable under UV light. I thought of heating at 75℃ for 5-10 minutes to inactivate DNase, will the heating affect MES buffer properties?
Probably your best approach is to use a fresh bottle of MES, keep isolated from general use, don't use a spatula and treat as you would reagents for RNA work and make your solution with nuclease free water. The likelihood of introducing DNAses is extremely low (RNase is much more problematic).
The MES buffer may turn slightly yellow when heated (although this may be insignificant after only 5-10 min at 75oC), but it may still be OK for your purpose. I'm not sure whether that treatment will permanently inactivate DNase. Avoiding DNase contamination in the first place, as suggested by Michael J. Benedik, is the best approach.
EDTA will affect your enzyme only if its activity is dependent on divalent metal ions.
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