correlating fluorimetry and spectrometry for DNA quantitation - (May/18/2006 )
I do my DNA quantitation both with spectrometry and with fluorimetry using the pico green.
I can never correlate these results.
For instance the DNA isolated from pecan seeds with nucleospin food kit is 241 ng/microl using spectrometry but 15 ng/microl using fluorimetry.
The OD260/OD280 ratio is 2.04, which suggests some RNA contamination. Can this attribute for such a huge difference between the two results ? There is the RNAseA digestion step in the isolation procedure, but RNAseA is added together with proteinase K, so maybe proteinase partly destroys RNAse...
To give a complete information, I use commercial salmon sperm DNA to calibrate picogreen fluorimetry.
Can anybody share their experience on (not) correlating these two DNA quantitation methods ?
Another question -
can anybody give me a simple protocol for RNAseA treatment AFTER the DNA isolation and for cleaning up the sample again ?
I don't have specific experience with what you are doing, but I just wanted to point out that A260 is a pretty non-specific measurement, and it would definintely be exaggerated if you had RNA contamination of your DNA.
For my own work, I have also used 2 different methods to measure RNA. Salts, phenol, and DNA can also absorb at A260, so when I have contaminated samples, my A260 reading is significantly higher than the reading I get from the bioanalyzer. Only when I clean up my RNA will the two readings become similar.
I hope that helps some!
thank you. You confirmed my fears that I will have to do more RNAse treatment.
I appreciate yor response.