OD260 and free dNTP - (Nov/12/2005 )
This may be a rather naive question, but does anyone know FOR SURE whether free nucleotides add anything to the OD260 measurements? Or is the absorbance at 260nm specific to the polymer (i.e. DNA)?
No, free nucleotides will definitely contribute to your A260 readings. Absorbance at 260nm is basically used to detect the aromatic rings that comprise the bases of a DNA fragment. Since free dNTP's also contain such rings they will influence your A260.
You could ethanol precipitate your sample to remove free dNTPs if needed.
Great. That's what I thought.
yes, free dNTPs add very much to your OD at 260 nm. The best way to test it is take OD of 10mM dNTPs used in PCR , it will give a very high OD around 0.8 at 260nm. It is due to the hyperchromic shift , since free or denatured and small strands of DNA give high OD than long and double stranded DNA