Recently I performed a DNA extraction using Qiagen DNeasy kit and the results show a 260/230 ratio of 1.05 likely due the presence of phenol or other organic contaminant... I have read that you can improve the 260/230 ratio performing the washing step twice. Does anyone has another advice?
You can do a precipitation of your DNA that has the low 260/230 ratio. That should take care of the contaminants.
Hi Hector. This is the protocol I have used and it works well:
https://projects.iq.harvard.edu/files/hlalab/files/ethanol-precipitation-of-rna_hla.pdf
Also, please consider that QIAGEN buffer AE used in the last step of DNeasy kit contains Tris that affects the concentration and absorbance values. Take a look at the link below:
https://www.neb.com/-/media/catalog/application-notes/mvs_analysis_of_na_concentration_and_purity.pdf?la=en&rev=be7c8e19f4d34e558527496ea51623dc
It's likely left over guanidine hydrochloride from buffer AL and AW1. Guanidine HCl is a chaotropic agent used to denature proteins, it's usually at a high concentration in the first wash buffer of most silica column kits, its purpose is to remove any protein that might have bound to the membrane with the DNA while not affecting the DNA much. It also absorbs pretty strongly at 230 nm.
So yes, washing twice with the desalting buffer (AW2 in this case) can help. Also consider leaving the desalting buffer on the column for longer time periods before centrifuging (4-5 minutes), that often reduces it significantly. If you're really worried next time, you can do both. In my experience as long as the desalting buffer isn't really old, doing multiple/longer washes with it doesn't appear to reduce yield and results in a much cleaner prep. Most desalting buffers in commercial kits are just 80ish% ethanol with a buffer (usually 10 mM Tris, pH 7.5-8), large dsDNA isn't going to dissolve off a silica column in the presence of 80% ethanol.
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