We are seeing decreases in the %'age of transfected cells (HeLa, COS7, MEFs) with time of storage over a few weeks, of purified plasmids at 4C. Seems more stable when stored at -80C. I don't think nicking is responsible as we don't see obvious changes in DNA migration in agarose gels. Anybody have any ideas what might cause this or how to decrease losses? We use kits (Qiagen mostly) to purify DNAs and elute with water or elution buffer (10 mM Tris).
I don't know for sure but I do know that as DNA is stored over time, it gets harder to get it back into solution so perhaps there are microaggregates. When we use DNA that has been stored a while we usually incubate it 5-10minutes at 37degrees C and then suspend up and down well before using. Only a guess...
I'm not sure if the rates in solution would be significant enough to cause the effect you're seeing, but the plasmids could be slowly accumulating base damage which prevents proper expression of the plasmid's markers. Treating the plasmids with a glycosylase/AP lyase would convert the base damage into nicks and allow them to be visualized as a gel mobility shift. Nth (endonuclease III) can be used for oxidized pyrimidines and abasic sites; Ung (uracil-DNA N-glycosylase) could be added with Nth to additionally catch deaminated cytosines.
Plasmids are more stable in EB vs. water. But why not freeze it? I never leave plasmids at 4C, and aliquot if I'm worried about freeze/thawing.
I agree that freezing makes sense for long term storage. But these are plasmids that used every few days or maybe once every couple of weeks. I thought about the EB vs water issue, though DNA sequencing companies prefer water as the TE can interfere with their reactions. As a result we have migrated to elution with water but maybe time to compare. Thanks for your answer.
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