Degraded DNA from Mouse Tissues - (Jul/11/2012 )
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?
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.
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?
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
First, thanks to those who have already assisted me in this issue. However, I'm still having problems and can't pinpoint the reason why.
Does anyone out there have experience with isolating DNA from mouse tissue? I'm still having degradation problems and can't pinpoint the reason. Once I isolate tissue, I freeze it immediately in liquid nitrogen, then keep it on dry ice, followed by crushing the tissue with a hammer. The minced tissue is then incubated overnight (50 deg, shaking incubator) in standard mouse tissue digestion buffer, which includes proteinase K and RNase A. The next day, the sample is mixed in phenol chloroform and spooled in isopropanol for isolation.
I'm not sure where the degradation is happening. I always thought that once the tissue is on dry ice, all nuclease activity is "dead," and then anything else becomes suppressed by Proteinase K in the overnight digestion.
Does anyone have any suggestions? Thanks!
Try shortening the RNase step - only an hour should work. I would do it before the PC extraction. Rnases are not very specific and so could be digesting your DNA partially with the long incubation.