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OD260 for DNA is minus! - (Nov/20/2002 )

Hi, I got very strang result when I assay the OD 260 of plasmid DNA, it was minus, like -0.009. -0.135. but run the same sample 2 ul on gel of 0.7%( the plasmid is about 4788 bp), there is bright band, much more brighter than the marker ( 50 ng ), I have not much knowledge about the machine used to assay the OD. Can anybody give me a hand to find out the problem. I need to accurately quantity the plasmid DNA concentration, which will be used for caculate the amount of DNA for transfection.

Looking forward your professional comment.

Thank you in advance.



Maybe your DNA is too less concentrated to measure....

Let's assume your band in the gel looks like it's about 200ng. So you got a concentration of 100ng/Ál. That dilluted 1:100 for OD260 measuring would give you a absorbtion value of about 0.02. Such low values don't go together with most photometers, let alone the precision of the value.

Another thing could be responsible for you measurements, maybe in combination with the one above: you always MUST measure against a correct control, i.e. if you're taking you DNA eluatet in e.g. Tris/EDTA and dillute it 1:100, that would be 1Ál DNA + 99Ál H2O. So your reference has to be 1Ál Tris/EDTA + 99Ál H20.

Hope I could help.


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