Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Nanodrop - (Nov/03/2012 )

Hi again,

What should be the ideal 260/280 and 260/230 ratio when analyzing samples on nanordrop?

-Mad researcher-

http://www.google.co.in/url?sa=t&rct=j&q=260%2F230+ratio&source=web&cd=1&cad=rja&ved=0CCIQFjAA&url=http%3A%2F%2Fbatzerlab.lsu.edu%2Fgenomics%2Fdocumentation%2F3130_NanoDrop_tips.pdf&ei=Tk6VUI6fHoSsrAeeqIDIDw&usg=AFQjCNEMyMURexSZJ6WXtZbkjNBtTHfNRw

-Inbox-

I referred to that article in past but it doesn't say anything specific about DNA. It just says 260/230 ratio should be around 2.0.
And from my past experience they say anything around 1.0 for 260/230 is pure.

So am confused now

-Mad researcher-

At 230 nm several organic compounds absorb and there are some important ones such as phenol, chaotropic salts or some peptides.
Anyway I won't put too much importance in these ratios as they're a rough indication that something might be wrong, or everything might be okay. E.g there are enough inhibitors that won't be included in this measures.

-hobglobin-

The 260:280 ratio should be 1.8 for DNA and 1.9 for RNA. However, as I found out recently, the ratios thing comes from a paper published in the 1940's which was using abs at 260 and 280 to measure the contamination of PROTEIN with DNA - and found that the absorbance at those wavelengths was good for doing so, however, the converse is not true- protein contamination of DNA samples only slightly affects the ratio of 260:280. Molecular Cloning: a laboratory manual (Sambrook et al) has more details in the more recent editions.

-bob1-

So, i shouldn't worry abou the 260/230 ratio? My 260/280 ratio is fine, i was worried about the 260/230 ratio

-Mad researcher-

I'd not care too much about it...it's a rough indicator for a clean or not clean sample....and it depends what you want to do afterwards, i.e. how clean your DNA has to be. My PCRs work with quite dirty DNA (mostly proteins) and I don't do Phenol/Chloroform DNA extractions before, so I don't have to care about Phenol contamination.

BTW on my Eppendorf Biophotometer manual they say the value of 260/230 should be larger than 2.0 for clean DNA and RNA, smaller values indicate contamination with salts, organic solvents or carbohydrates. (Just as addendum to my previous post).

-hobglobin-

Thanks hobglobin. I don't use Phenol chloroform for DNA extraction. i usually use a kit from Sigma aldrich (mammalian genomic DNA extraction kit).

Does, the eppendorf biophotometer manual say anything about the 260/280 ratio?

-Mad researcher-

Yes the same as bob1 mentioned. With clean DNA the ration is 1.8, clean RNA 2.0. Smaller values indicate protein and/or phenol contaminations. But consider the limitations bob1 mentioned.

-hobglobin-