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Disparity between nanodrop and gel

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#1 cdarwin



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Posted 15 July 2014 - 01:53 PM

Hi everyone, 


So I have scoured the boards trying to find someone else who had had a similar issue, but I came up empty-handed. 


I am doing simple phenol chloroform extractions with EtOH precipitation. My yields are pretty good for environmental samples and I was pleased, until today. 


I decided nanodrop the positive control that shows up on gel after PCR (as you would hope), and got some really strange results: the numbers were very similar to those of the negative control. 


negative: 2.07 (260/280) 0.10 (260/230) 1.6 (ng/ul) 

positive: 1.81 (260/280) 0.03 (260/230) 1.2 (ng/ul) 

my sample: 1.94 (260/280) 0.41 (260/230) 82.5 (ng/ul)        (I know that the second ratio is way low, but I can fix that)


Last time I nanodropped (verb?) the positive in September, the numbers were very similar to my current sample (what you would expect).


The PCR blanks come up negative, so I know I'm not having a contamination problem. I am blanking the Nanodrop with the correct solvent. So why would DNA show up on a gel and not on a Nanodrop? (The reverse of the normal problem.) 


Thank you so much for your help! 





#2 bob1


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Posted 15 July 2014 - 05:26 PM

I'm nt quite sure here whether you are measuring the concentration of the PCR or if you are measuring the concentration of DNA used as template for a PCR?

#3 Trof


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Posted 16 July 2014 - 09:10 AM

cdarwin: You are not showing the absorbance values of Nanodrop, only ratios (which doesn't tell anything) and calculated concentration. It's difficult to tell anything about it. Also Nanodrop gives a full spectrum, that would be also important.

It can be anything from a simple fail of single measurement to the fact, that something is wrong with your samples.


Not only, that you may have you positive control negative actualy, but as you are talking enviromental samples, the "negative" may actually contain a lot's of DNA, but also inhibitors that make PCR output negative (this could be tested).


Anyway, I'm not really sure, why you measure that in the first place. You need the samples to amplify? If so, you don't need nanodrop at all, DNA is amplifyable way beyond the lower limit of Nanodrop detection.

If you need to check your control sample, you can run it on gel, if it's concentrated enough.


Absorbance measurements are really only usefull if you have a very clean product, for concentration calculations. I would say it's much more useless with enviromental samples than with some more homogenous sources. I'm affraid phenol/chlorophorm extration can't ged rid of all PCR contaminats, that may block the reaction, and since it's enviromental, it's hard to say when your samples does contain inhibitors and when it doesn't.

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