Hi,
Having an odd experience with some DNA samples extracted from chicken feces. The DNA was first extracted from my samples using the Powersoil kit. I then treated these samples with a 1/10 concentration of RNase Cocktail Enzyme Mix at 37 degrees C for 1 hour then performed phenol:chloroform extraction on them. This was followed by ampure purification at 0.75X.
When I measure their concentration by nanodrop I sometimes get 260/280 values of 1.85-1.92. Ideally I want 1.8, to show that I have pure DNA. My 260/230 values are >2.2 and the absorbance curves look good, not showing any signs of contamination.
Oddly, when I use the Qubit HS dsDNA assay, I get the same concentration values (~110ng/ul) as I get from the nanodrop. The qubit assay is specific for dsDNA, thereby indicating that all of the nucleotides I am measuring using the nanodrop are coming from DNA and there is therefore little to no RNA.
Has anyone got any suggestions as to why, if I have hardly any RNA in my samples, the 260/280 ratio is not 1.8?
Hi Laura ..
Instead, you should perform agarose gel electrophoresis to check the DNA integrity and investigate the presence of DNA smear.
It would be more conclusive than that looking at A260/280 ratio. Furthermore, a 260/280 of for DNA sample should be fine for most of the downstream applications.
'
follow these links
ISOLATION AND PURIFICATION OF GENOMIC DNA
http://envfor.nic.in/divisions/csurv/biosafety/Gef2/T5/16%20Dr%20Randhawa_%20Isolation%20&%20purification%20of%20genomic%20DNA.pdf
https://www.thermofisher.com/iq/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/ultraviolet-visible-visible-spectrophotometry-uv-vis-vis/uv-vis-vis-instruments/nanodrop-microvolume-spectrophotometers.html
Best Luck.
In the beginning, be sure to extract DNA and not to contaminate it,
High 260/280 immaculateness proportions are not demonstrative of an issue. In spite of the fact that virtue proportions and unearthly profiles are importantindicators of test quality, the best marker of DNA or RNAquality is usefulness in the downstream application ofinterest.If the immaculateness proportion is altogether higher thanexpected, it is best to audit the ghastly profile as a primarymeans of troubleshooting.It is critical to take note of that there are events when the purityratios are inside expected breaking points, yet there is an issue withthe test
http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
regards
You're DNA is likely fine. The Nanodrop is not a perfect system for differentiating between nucleic acid type. It is likely you still have trace amounts of protein or phenol in your sample that are providing some slight absorbance in the 280 range.
The corroboration of concentration between two different techniques is highly convincing that your DNA is decently pure.
https://www.biotek.fr/fr/resources/application-notes/low-volume-high-throughput-workflow-for-analysis-of-nucleic-acid-samples-for-biobanking/
Hi Laura ..
Instead, you should perform agarose gel electrophoresis to check the DNA integrity and investigate the presence of DNA smear.
It would be more conclusive than that looking at A260/280 ratio. Furthermore, a 260/280 of for DNA sample should be fine for most of the downstream applications.
'
follow these links
ISOLATION AND PURIFICATION OF GENOMIC DNA
http://envfor.nic.in/divisions/csurv/biosafety/Gef2/T5/16%20Dr%20Randhawa_%20Isolation%20&%20purification%20of%20genomic%20DNA.pdf
https://www.thermofisher.com/iq/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/ultraviolet-visible-visible-spectrophotometry-uv-vis-vis/uv-vis-vis-instruments/nanodrop-microvolume-spectrophotometers.html
Best Luck.
Hi Mohammed,
I had already checked my DNA on a gel and it gives a good clear band >65kb.
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