I'm doing DNA extraction using Chelex and before DNA purification, it have 260/280 ratio start from 1,1-1,4. Usually after DNA purification, 260/280 ratio will ranging between 1,8-2 (Pure DNA) but all of my purification result shows 260/280 ratio higher than 2 (between 2-2,5). But besides 260/280 ratio, everything seems normal (indicate that the purification performed well). I'm using ROCHE High Purification Kit for the DNA purification.
This can solve all your confusions regarding 260/280 and 260/230 ratio.
http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
unusual high rate 260/280 may be due to co-extraction of RNA in your DNA extraction system. Have you analyzed extracts on agarose gel electrophoresis?
what is 260/230 value of your samples? Is it also above 2? are you treating your DNA with RNAse? you should check your purified DNA on agarose gel, it will give you an idea what is happening with your samples.
To Cyril: What do you mean with co-extraction of RNA?
To Madiha: some of it are above 2 as well, but most of them ranging between 1-1,3.. I'm not using RNAse though...
Can you guys give some example about what happen? Since i didn't check my samples on agarose gel...
the contamination of RNA in the DNA extraction is frequently observed when in the method no RNAse traitment was applied. The ratio 260/280 must be appreciated with DNA only but not with a mix of DNA and RNA. In this case of the présence of DNA and RNA in your extraction you obtain a ratio 260/280 > at 1.8.
Check your samples as possible on agarose gel.
Better to use RNase during extraction and every time after DNA extraction check the quality by running it on Agarose gel. Good Luck.
It is a sign of RNA contamination. After purification digest your sample with RNase A. I suggest you using fastdigest buffer for this step.
260/ 280 ratio of ~1.8 is generally accepted as “pure” for DNA and a ratio of ~2.0 is generally accepted as “pure” for RNA.
For any DNA sample with A 260/280 ratio more than 1.8 indicates the presence of RNA as contamination.
It is always suggested to give RNAse treatment at the time of DNA extraction so as to get pure DNA sample.
Thanks all. This has been very helpful for me too. What is an acceptable 260/230 ration for RNA? Thank You.
This can solve all your confusions regarding 260/280 and 260/230 ratio.
http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
Another one that may be useful
http://biomedicalgenomics.org/RNA_quality_control.html
The five nucleotides that comprise DNA and RNA exhibit widely varying 260/280 ratios**. The following represent the 260/280
ratios estimated for each nucleotide if measured independently:
Guanine: 1.15
Adenine: 4.50
Cytosine: 1.51
Uracil: 4.00
Thymine: 1.47
https://www.researchgate.net/deref/http%3A%2F%2Fwww.bio.davidson.edu%2Fprojects%2Fgcat%2Fprotocols%2FNanoDrop_tip.pdf
I found lab protocol that states 260/280 ratio higher then 2 appears to be related to DNA degradation.
Hi Christine Dwi A P W ,
you wrote that all your Nanodrop results for the 260/280 are between 2 and 2.5.
2.5 seems a bit high, but if you have 2.0, 2.1 or maybe even 2.2, I wouldn't worry about it too much, unless you need extra pure DNA.
If you just want to amplify certain DNA regions, I would just go ahead with PCR (even the 260/280 ratios are a bit off). It will probably work. Just try PCR & gel electrophoresis, and check whether your amplicons yield good, crisp bands on the gel.
Having said this, 2.5 seems a bit high, and I would imagine that the 260/230 ratio is a bit off for that sample too. You may also want to consider redoing Nanodrop for that or all samples, making sure that the DNA/buffer solution is thoroughly thawed and mixed, and that you use about 2 ul in the Nanodrop. (I remember that when I first used Nanodrop, I was told 1 ul is enough, but I soon realized that 1 ul gave me very inconsistent results for the same sample).
I hope this helps!
Cheers, Oliver
I had this problem with my samples after gel purification and I found out that the responsible for it is Guanidine thiocyanate. This contaminant is present normally in the buffer for the gel dilution and absorbs at a high ratio the UV at 260 nm of wavelength, but doesn´t absorb the 280 nm wavelength. That is why, you can get a peak with a great concentration that seems like DNA but it is actually a contaminant of the method of purification. Normally this peak has it's max absorbance around 250 nm but if you are not paying much attention to the curve you could probably think is pure DNA.
The ratio 260/280 can rise even to values of 10-15 and you won't have much DNA. This is why is always good to run an agarose gel with the product of your purification in order to be sure that you have the band or bands of the expected molecular weight.
Cheers Matias
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