Protocol Online logo
Top : Forum Archives: : Molecular Biology

Problems with 260/280 ratio - Problems with 260/280 ratio (Aug/22/2008 )

I am getting negative numbers(-1.3) in my 260/280 ratio when I try to quantify my extraction of genomic DNA, does this just mean that it is completely contaminated with protein or is the result interpretable at all,

Thanks in advance for any advice


Do you only have a readout of the ratio or also the seperate values for 280 and 260? Perhaps you could get a better hint of what is the problem, if you can see the individual values. Is your blank o.k.? The same buffer as for your genomic DNA?


There is only the readout for the ratio 260/280 on our machine, have blanked it with the same nuclease free water that the DNA is eluted in, its very frustrating as I also sometimes get even lower values, down to -10 once.


You want your ration to be around 1.8

Without individual values I can't say whats wrong but either way, I wouldn't trust that DNA.


You might want to have a technician come and check out the machine, it might need to be calibrated.


I thought that too but other people use it and their results are normal, there are very low amounts of DNA in my sample, 2ug/ul, could that be the cause?


What do you mean by normal? What are they doing to get these normal results? What kind of cuvettes are they using vs. what you are using?

With lower concentration DNA elutes you want to use a smaller volume cuvette to obtain an OD so you dont have to dilute the sample as much. A low conc. sample would not cause a bad reading, not that I am aware of anyway. Make sure all readings are above a 1.0 to be reliable...ex: OD260 is 1.3


Do you really mean 2ug? Because that is not a low concentration at all. When you do plasmid preps you might have 500ng-1ug. At least in my experience. Or did you mean 2 ng? If that is the case than you won't be able to detect it with a spec anyway. Use an agarose gel and compare your probe to known concentrations. The minimum amount of DNA for detection with EtBr is 7ng.
You might wanna try that just to see if you have DNA at all