A260 too high - (Feb/25/2008 )

Hello everyone,

I have a question about plasmid prep. I extracted plasmid from 3ML of overnight culture following the standard alkaline lysis protocol. At last, I dissolved the pellet in 100ul of water. Then I measured A260 to calculate concentration of the plasmid. The reading of a 1:100 dilution is 0.7, which means the concentration of my plasmid is 0.7*100*50=3500ng/ul. That is impossible!!! A260/A280 is about 2. I just don't know what caused the misreading. I also ran the plasmid on a gel, I think the concentration is about 300~500ng/ul. Does anyone know the reason?

BTW, I have used phenol:chloroform in the process, and contaminated RNA has been digested with RNase.

Thanks for you help!

Could that be a phenol contamination? I just found that phenol has strong absorbance at 260nm.

You can extract your DNA with chloroform or ether to remove the phenol and then reprecipitate it.

thank you very much!