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RNase digestion problems

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RNase digestion problems

Postby justcurious91 » Mon Sep 27, 2010 2:46 am

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 _at_ 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
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Postby canalon » Mon Sep 27, 2010 7:01 pm

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?
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Re: RNase digestion problems

Postby justcurious91 » Mon Sep 27, 2010 7:50 pm

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?
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Postby canalon » Mon Sep 27, 2010 11:06 pm

What is the best hypothesis that would explain such a result?
What does disappear? What could have that effect?
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Re: RNase digestion problems

Postby justcurious91 » Wed Sep 29, 2010 5:06 am

maybe DNase contamination in the RNase since the DNA is also being digested. so right now i am preparing a fresh stock of RNase.
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Postby canalon » Wed Sep 29, 2010 11:41 am

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