I´m making some DNA gel extration for cloning and the 260/280 ratio is pretty high. Does anyone know what it means or how to get better ratios? I´m using a gel extraction kit from invitrogen and I´m running my DNA on a low melting agarose.
Hi,
The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as
“pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate
the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
Celine
What does it mean pretty high?
Maybe it will help you
http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
Are you having problems with your subsequent experiments? If not I wouldn't worry too much - gel extractions can give odd spec results, a peak at 230 from ethanol or other solvents is quite common. From my experience, sometimes you even get different peaks depending on which gel stain you used (for example EtBr vs GelRed)
it should be 1.7-1.9, if cloning is OK, you don't need to do anything just for better numbers, but if downstream applications are problematic, you can re-purify your samples with some commercial kits or simpler recipe as follows:
1. dilute your sample 3 times
2. add 1 volume of chloroform (equal to your sample after step1), mix gently
3. centrifuge 10 000 rpm
4. transfer supernatant to a new tube and add 1/10 volume of 3 M sodium acetate, mix gently
5. now add 3 volumes of cold 96 % ethanol, mix and keep at -20C for 40 min, if you want more pellet - leave for the night
6. centrifuge at 12000 rpm for 6 min
7. remove liquid, add 2 volumes of 70 % ethanol, keep at -20C for 20 min
8. centrifuge at 12000 rpm for 6 min
9. remove alcohol, dry pellet completely, resuspend in your DNA buffer (TE, H2O)
during re-purification you will lose some DNA, but you can dilute in a smaller final volume than was before purification
I am also having this problem. I am not sure if I am just interpreting my data wrong, but my results were:
Nucleic acid concentration: 1.7 ng/uL
A260: 0.035
A280: 0.005
260/280: 7.44
260/230: 0.05
Sample type: DNA
Factor: 50.00
The doctoral student that was helping me run the nanodrop said my sample was fine and fairly pure, but I am not sure how he came to that conclusion given the 260/280 ratio.
@Kathrine If you get low nucleic acid concentrations like 1.7 ng/uL the indicated ratios are hard to interpret. That should tell you also the Nanodrop while measuring.
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
High 260/280 purity ratios are not necessarily indicative of a problem. However, a very high ratio can suggest a poor quality blank eliminating too much signal near the 280 nm wavelength.
Is there a chance of any technical malfunction with the nanodrop instrument? I have had such an issue too, where the instrument reads an abnormally high ratio but the RNA quality was excellent as seen from gel electrophoresis. Moreover, the sample had no problem in any of the downstream procedures. But the question and doubt still remain!
Is a ratio of 2.2 for 260/230 high? If I do have EtOH contamination, how do I get around it?
I get a value of 3.3. I extract my DNA with Trizol, choloroform, supernatant ethanol precipitation. The detailed data is as following:
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