3 replies to this topic

[hotstuffdb22]

Hello,
I'm struggling with isolating DNA from mouse tissues.  About 3 out 10 DNA samples each time show a smear on a 1% gel.   My end goal is a southern blot.  For isolation, I mince the tissue into small pieces and then an overnight proteinase K at +55 deg, rotating 1000 rpm.  The next day, I do a standard phenol-chloroform extraction followed by a 4-6 hour RNase incubation (+55), and then another phenol-chloform extraction, followed by a final ethanol precipitation.  My first reaction to the presence of degraded samples was nuclease activity, but not all the samples are degraded, so this doesn't appear to be the issue.  Does anyone have any suggestions or protocol recommendations to overcome this hurdle?

Thanks!!!

[Curtis]

I have had similar problems and they were because of RNA contamination. I have a feeling there is a problem during your RNase incubation. Why don't you add RNase in the last step? just add it to water.

[hotstuffdb22]

Thanks a lot for this suggestion!  It makes intuitive sense, but does leaving my final DNA in a solution with RNase have any downstream effects on assays performed?

[Curtis]

sorry for late reply. No not really. It shouldn't be a problem. We use our DNA samples usually in PCR or digestion/cloning. addition of RNase doesn't really cause a problem. Infact, most protocols say that you can inactivate RNase by heating your sample. But usually RNases are really badass I have read a lot that they don't really get inactivated even after autoclave st 120C. ...just don't worry