RNase digestion problems

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    • #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

    • #101518
      canalon
      Participant

      Controls are your friend:
      Did you have DNA in the tube in the first place (saving a few ┬Ál 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?

    • #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?

    • #101522
      canalon
      Participant

      What is the best hypothesis that would explain such a result?
      What does disappear? What could have that effect?

    • #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.

    • #101559
      canalon
      Participant

      Exactly my conclusion, and what I would have done. Good luck

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