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DNase treatment from RNA using expired RQ1


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#1 kutayozturk

kutayozturk

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Posted 04 April 2012 - 05:59 AM

Hi,

I have a question regarding with RNAs again. I have plenty of different RNA samples ready for qRT-PCR analysis. For that, I need to do DNase treatment using Promega RQ1 Dnase. Our DNase is very very old, expired in 2001. I think it still works a bit, since I managed to get cDNAs and one qRT-PCR data. However, the main problem is that the concentration gets very low (estimated 150ng/uL, obtained 30 ng/uL- No idea where did all the RNA go) and I awkwardly gain high 260/230 absorbance ratio (my RNA samples looked excellent before hand). I am not sure if I can do LiCl precipitation, because my total RNA concentrations are around 120-200 ng/uL. One is even 80ng/uL. I have Turbo DNase from Ambion mMessage mMachine in vitro transcription kit, and LiCl, too. So, if you suggest me to do LiCl precipitation, I will do by using it. The protocol in the web tells me how to do this part just after in vitro transcription, so the amount of RNA added will be different in my case. What do you suggest me to do?

Thanks,
Kutay




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