I have extracted DNA oligonucleotide of size 77 bp from a crushed 10% PAGE gel using 3 rounds of LiClO4 in water. The extracted elute was dried down in a vacuum centrifuge to a certain volume and then precipitated with 5 volumes of Ethanol before taking a spec on a NanoDrop. I do have a significant peak, characteristic of a DNA oligonucleotide, at 260 nm with a A260/A280 ≃ 1.75.
However, I also get a peak at 380 nm. I used to get this since a long time but I could not understand what it meant (did not find any literature reference). I do not want to worry about it too much, as long as I have my oligo, but I would like to know what it is. A colleague of mine also got the same peak, though not very prominent, who did not do a PAGE purification but direct UV Spec after Phenol/Chloroform extraction and LiClO4 in acetone precipitation.
What would you suggest?
Thank you.
this link useful
bio.lonza.com/.../Lonza_BenchGuides_SourceBook_Section_IV_-_D...
regards
Did you blank (zero) your spectrometer with water/tris ?. 380 absorption could be anthracene or polycyclic compounds and I wonder if your ethanol has an organic contaminant. Try measuring the absorbance of your ethanol against a water/tris blank to see if the peak appears
260/280 ratio 1.75 shows that the quality of your nucleic acid is not good. It may be some sort of phenolic contamination. Try washing with 70% ethanol 1 or 2 times.
Also I am agree with Paul's suggestion.
Nilay
You have extracted DNA from page but what about it's original source?
Anyway washing can solve your problem most of the times. try it.
Regards
Nilay
usually, we precipitate the oligo directly after extraction from PAGE (TE-NaCl buffer), with sodium acetate 3M and 3 vol absolute ethanol. Did your oligo migrate as one of the loading buffer marker (xylene cyanol or bromophenol blue)? Sodium acetate/ethanol precipitation prevents markers precipitation. I don't know about LiClO4 extraction/speedvac/ethanol protocol.
Best regards
Laurent
- Badges
- Science method