I am trying to remove RNA contamination from my DNA samples. I gave RNAse treatment for DNA resuspended in 50 ul of TE buffer. After 1 hour of treatment I reprecipitated the DNA using 1/10 V of Sodium acetate and 2.5 V of 100% Ethanol.
Finally I resuspended the DNA in low salt TE buffer (10 mM Tris, 0.1 mM EDTA). I observed that the concentration of the sample dropped by 4 times and also the 260/230 declined from 1.8 to 0.9
Can anyone explain whats happening ?
Regards
Gurpreet
Dear Gurpreet
I can assume several reasons:
1) Your RNAse worked very well. You just had a lot of RNA contamination of the DNA extracted. If You used Nanodrop, it cannot distinguish between DNA and RNA. So Your first measurement gave You a total concentration of nucleic acids in the sample. After RNAse treatment - only DNA. And it was much lower.
2) Your RNAse was contaminated with DNAse. In this case, after treatment You will have just nucleotides.
3) Rarer reasons which I would think about after excluding firsts two.
The drop in 260/230 can be explained by simple decrease (reason 1) or total absence (reason 2) of DNA. Specifically, if You got very low (close to noise) optical absorbance at 260. Also, since You re-precipitate DNA after RNAse treatment, You used salt again, which is one of the causes of high absorbance on 230, so I would clean the pellet before TE dissolving in 70% EtOH (twice).
What kind of extraction method did You use?
this is quite expected due complxes formed from DNA and RNAse enzyme. however it should not affect concentration too much, you can check by electriphoresis
This is quite expected due complxes formed from DNA and RNAse enzyme
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