RNase digestion problems

last updated by

canalon 14 years, 2 months ago

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- September 27, 2010 at 2:46 am #13828
  justcurious91
  Participant
  hi
  i have run into a few problems in the experiment for plasmid extraction. i did all the steps for boiling lysis followed by phenol-chloroform protocol. for two tubes,i also gave an additional RNase digestion (incubation time 1hr @ 37C). during electrophoresis only the samples with phenol-chloroform treatment had the proper bands while the ones with phenol chloroform+RNase didn’t show any bands at all. can anybody help me interpret these observations?
  thanks
- September 27, 2010 at 7:01 pm #101518
  canalon
  Participant
  Controls are your friend:
  Did you have DNA in the tube in the first place (saving a few µl before digestion would have told you that)?
  Is your RNAse not degrading DNA (test with a solution that you know contains DNA) because of a contamination?
- September 27, 2010 at 7:50 pm #101519
  justcurious91
  Participant
  ya i ran a few controls and observed that everything, even the standards when treated with RNase were showing up as a blank. i think there is some contamination in the RNase but can you explain why the entire lane is appearing as a blank?
- September 27, 2010 at 11:06 pm #101522
  canalon
  Participant
  What is the best hypothesis that would explain such a result?
  What does disappear? What could have that effect?
- September 29, 2010 at 5:06 am #101549
  justcurious91
  Participant
  maybe DNase contamination in the RNase since the DNA is also being digested. so right now i am preparing a fresh stock of RNase.
- September 29, 2010 at 11:41 am #101559
  canalon
  Participant
  Exactly my conclusion, and what I would have done. Good luck
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