Protocol Online logo
Top : Forum Archives: : Molecular Biology

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)?

-namekuji-

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.

-Hank

-haringsh-

Great. That's what I thought.
Thank you!

-namekuji-

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

-Shashi Prakash Singh-