Checking DNA quality with 260:280 ratio - (Sep/03/2004 )
I routinely use silica membrane spin columns and chaotropic salts to purify DNA from plasmids or after PCR. I then check the yield with a spectrophotometer. I've noticed that the 260:280 ratio is almost always greater than 2.0; occasionally it is as high as 3. Does anyone know why this is? The reading I've done suggests that high ratios are indicative of contamination with phenol or chloroform, but that isn't possible here. Could it be an artifact caused by diluting the DNA in TE to measure the concentration? I use anywhere from 2-5 ul of DNA in 50-60 ul of TE in the cuvette.
Make sure your 260 reading is below 1.000 and above background levels of your spec. If the reading is high at 260, it tends to be not as accurate, which would throw off your ratio.