My plasmid purity changes with different dilutions - (Apr/11/2006 )
After plasmid prep, I checked the plasmid purity. I checked in 2 dilutions. One is dilute 1ul plasmid DNA in 99ul milliQ water, and get a A260:A280 ratio of 1.3. The other one is dilute 5ul plasmid DNA in 95ul milliQ water, and get a A260:A280 ratio of 1.7.
I would think the A260:A280 ratio shouldn't be affected by dilution factor. Is there any possible problem with my plasmid DNA?
And which purity data should I trust? 1.3 or 1.7?
-Ohmyhead-
QUOTE (Ohmyhead @ Apr 11 2006, 07:53 PM)
After plasmid prep, I checked the plasmid purity. I checked in 2 dilutions. One is dilute 1ul plasmid DNA in 99ul milliQ water, and get a A260:A280 ratio of 1.3. The other one is dilute 5ul plasmid DNA in 95ul milliQ water, and get a A260:A280 ratio of 1.7.
I would think the A260:A280 ratio shouldn't be affected by dilution factor. Is there any possible problem with my plasmid DNA?
And which purity data should I trust? 1.3 or 1.7?
I would think the A260:A280 ratio shouldn't be affected by dilution factor. Is there any possible problem with my plasmid DNA?
And which purity data should I trust? 1.3 or 1.7?
what are your actual readings? if they are too low or too high then you will get invalid results.
the general rule of thumb for linearity (at least for older spectrophotometers) is od >=0.1 <=0.7.
i'm sure some will argue that you can rely on higher readings, and that may be so, but i wouldn't rely on values much lower.
-mdfenko-