I have done DNA extractions with a good quality kit and have got a fair concentration of DNA but its purity is really very low as A260/280 ratio is 1.45. Is there any way to get rid of contaminations without losing much DNA?
ethanol precipitation after a regular extraction can improve your DNA quality. Also depends of your tissues. If you are extracting DNA for example from invertebrates methods like CTAB can improve your DNA yield and quality. Make sure to use the right method depending on your sample.
In my experience, Zymo Research Fecal DNA MiniKit regularly gives those ratios, and the DNA is still PCRable. We've sent those in for sequencing regularly.
A comment: NanoDrop is not a super reliable method for DNA quantification (Qubit is reliable), so I never use the NanoDrop anymore for kits. If your downstream application isn't too expensive, just go on and see if it works
Hi there!
The A260/280 ratio of 1.45 indicates protein contamination. Check your A260/230 and if it is in the range of 2-2.5 then it is not any organic solvent (phenol or chloroform). To remove proteins I have used Phenol-chloroform-isoamyl alcohol (PCI) which gives quite a good yield. The only precaution to take is dilute your DNA with TE and leave a good amount of the aqueous phase (as DNA is diluted there won't be much loss) making sure that you don't accidentally take phenol or chloroform into the DNA solution. To remove residual PCI (if any at all) you can heat the solution for 5 mins at 65 degree, let it cool slowly while the residue evaporates and then do ethanol precipitation (3M sodium acetate and 95% ethanol) overnight at -20 or 1 hour at -80 and then give a thorough wash with 70% ethanol. Let the DNA pellet dry for sufficient time and dissolve it in TE and use it.
If you are going to use the DNA for PCR then it might not be an issue, but if you intend to transfection with the DNA contaminating with protein, it'll unnecessarily cause a lot of cell death and affect your subsequent experiments. Depending on the downstream process, you might want to decide the purification step.
Hope it helps!
The Zymo Clean & Concentrator kit might help. I used their RNA version recently and it improved the ratios quite a bit (in our samples it was the 260/230 that was low), and I estimate we recovered ~85% of the sample. You could ask your distributor if they have trial kits so you could test it on a few samples. I also agree with the others who said if you're using the DNA for PCR I wouldn't worry about it. Good luck!
me to , they said me that NanoDrop is not a reliable method for DNA clearness . so i am waiting primers to certify the extraction purity .
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