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help interpreting this data


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#1 molbio1234

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Posted 02 April 2014 - 05:21 AM

I have Nanodrop reading and need help knowing if DNA is pure or not. I need a second opinion.

 

The 260/280 reading is 2.17. I have seen places say 1.8 is ideal for DNA 260/280 purity. I have seen some other places go from 1.8-2.0. I believe that 2.17, there could be possible protein contaminants.

 

The 260/230 is 3.15. Which is higher than the normal 2.0-2.2 range. I read the DNA in triplicates, and the lowest of the triplicates for 260/230 was 2.65. Does it mean it there is a lot of chemical contaminants? What type of chemical will give a reading like this, between 2.65-3.15? I blanked with water and DNA buffer, then measured each as negative controls. They all read 0, so they are both at baseline.

 



#2 bob1

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Posted 02 April 2014 - 11:51 AM

The 260:280 thing is dependent on the solvent - in water 1.8 is the measurement you would get with pure DNA - however, RNA is a very very similar molecule so slight contamination with this would also give you a reading of 1.8.  If you go and look at the literature, you will find that actually the 260:280 ratio is a good method of measuring the contamination of PROTEIN solutions with DNA,but not the other way around, where you can have quite a large amount of protein in a DNA solution without the protein affecting the 260:280....  basically take these measurements with a healthy skepticism.

 

The best thing to do is look at the curves generated - if they are nice smooth curves with no peaks in the 230 range, then you are probably fine.



#3 mdfenko

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Posted 03 April 2014 - 04:35 AM

also, what are the actual readings? if they are very low then the ratios may appear to be abnormally low or high (small changes in absorbance will make large changes in ratio).


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