Accuracy of Nanodrop to measure plasmid concentration (View forum version)



science noob

Posted 09 October 2012 - 03:12 PM

Does anyone know the accuracy of using Nanodrop to measure DNA content after a plasmid prep? Some people stand by it whereas some prefer to run a gel to check if it "matches" up with the concentration.

I also know that it's important to check the 260/280 absorbances ratio in the Nanodrop for purity. Accuracy?

bob1

Posted 09 October 2012 - 06:51 PM

It is as accurate a method as you will get with UV spectroscopy. The point about also running a gel is that you can get an idea of the integrity of the DNA and potential contamination with RNA, genomic DNA and degraded DNA.

The 260:280 ratio tells you about the purity of the DNA 1.8 for pure DNA, 1.9 for pure RNA, but these values are altered by the solvent and to some extent affected by phenolic and protein contamination.

science noob

Posted 09 October 2012 - 08:28 PM

It is as accurate a method as you will get with UV spectroscopy. The point about also running a gel is that you can get an idea of the integrity of the DNA and potential contamination with RNA, genomic DNA and degraded DNA.

The 260:280 ratio tells you about the purity of the DNA 1.8 for pure DNA, 1.9 for pure RNA, but these values are altered by the solvent and to some extent affected by phenolic and protein contamination.


So does that mean if my plasmid prep product had a 260:280 ratio of 1.9, it is most likely 'contaminated' with RNA?

bob1

Posted 10 October 2012 - 12:14 AM

Not necessarily - what did you resuspend it in?

science noob

Posted 10 October 2012 - 12:24 AM

Not necessarily - what did you resuspend it in?


H2O.

bob1

Posted 10 October 2012 - 12:49 PM

Here's what Sambrook et al. (Molecular cloning: a laboratory manual) say: "The specific absorption coefficients of both DNA and RNA are affected by the ionic strength and the pH of the solution. Accurate measurements of concentration can only be made when the pH is carefully controlled and the ionic strength of the solution is low"

So measuring in H2O is not a good start point for determining the ratio - you need to be in a buffered solution of some sort.

They also (and this is new to me too) have this to say about the A260:A280 ratio (paraphrased a bit): For many years the ratio of absorbance at 260 and 280 has been used as a measure of purity of DNA and RNA. This method comes from a paper in 1942 that shows that the ratio is a good indicator of contamination of protein samples by nucleic acids. The reverse is not true! DNA and RNA absorb so strongly at 260 and 280 that the presence of protein doesn't change the ratio much.
They then have a table demonstrating this.

Trof

Posted 10 October 2012 - 01:24 PM

RNA has in TE buffer ratio more about 2.