RNase digestion problems

[justcurious91]

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

[canalon]

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?

[justcurious91]

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?

[canalon]

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

[justcurious91]

maybe DNase contamination in the RNase since the DNA is also being digested. so right now i am preparing a fresh stock of RNase.

[canalon]

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

[Author]

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