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Help- weird RNA concentrations after DNase treatment

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#1 Alleria

Alleria

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Posted 21 April 2014 - 04:33 PM

Hi all,

 

I used TURBO DNase I (Ambion) to treat my RNA samples and then extracted the RNA with phenol:chloroform followed by ethanol precipitation. During the spin/wash step (20min, 13,000rpm), I noticed that my pellets didn't stick tightly to the bottom of the tube and looked very "fragile" (not like the nice pellet that I got before DNase treatment). In addition, when I measured my RNA samples using a spec, I got really weird concentrations. Some were incredibly low and some were even higher than the original amount of RNA before treatment. I also measured them multiple times and my concentrations were not consistent (eg, 1st time 400ng/ul, 2nd 890ng/ul, 3rd 1049ng/ul).

 

Is it because my RNA samples are not pure? But I have done one phenol extraction/precipitation after DNase treatment. Should I do two times phenol:chloroform extraction?

 

Please help! Any thoughts would be much appreciated!

 

No need to double post. I've deleted your other one.  Bob.







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