Dear colleagues,
I am extracting a genomic DNA for sequencing with nanopore, and therefore am very cautious about quality checks. Recently I eluted the same DNA sample from a purification column with water and with elution buffer (EB), because, apparently, water leaves half of the DNA on the column, so I added EB to be sure that I got it all.
Next, nanodrop ratios were good for water solution but for the DNA in EB 260/230 ratio was 8. I did blank against the buffer, when measuring it.
What can be the reason and should I worry about the quality of my DNA in EB?
The EB was supplied with the column an is standard Tris-EDTA (DNA clean&concentrator kit from Zymoresearch).
Looking forward to your advice!
Hi Dr. Victoria Shabardina
260/230 ratio is used as a secondary method of nucleic acid purity. The common range for a pure sample is considered as 2.0-2.2. If the ratios are lower or abnormally higher it indicates the contamination of the samples with certain protein or phenolic compounds. A contaminant that absorbs around 260 nm other than DNA may also cause the problem. Moreover, higher ratios might have resulted due to errors in blank measurement. Maybe an inappropriate solution was used for the blank measurement. The blank solution should be the same pH and of similar ionic strength as the sample solution. If you clean your DNA product with 70 % ethanol well and check again, I think the problem will be solved.
I think this document will help you if you are using a nanodrop.
https://biosci-batzerlab.biology.lsu.edu/Genomics/documentation/vendor/3130_NanoDrop_tips.pdf
Some contaminants have characteristic profiles, e.g. phenol, however many contaminants present similar characteristics: absorbance at 230 nm or less. Abnormal 260/230 values may indicate a problem with the sample or with the extraction procedure, so it is important to consider both.
A high A260/A230 ratio may be the result of • Making a Blank measurement on a dirty pedestal • Using an inappropriate solution for the blank measurement. The blank solution should be the same pH and of similar ionic strength as the sample solution.
Sandhya Jayasekara
Hi Sandhya, the thing is that I eluted the same DNA prep with water an with the buffer. And the water solution has good values. Only the buffer solution showed this 8. Including, that the ration before the column was good also (2,2). So, something happened in the buffer, but what?.. I did blank Nanodrop accordingly and clean.
Hi again Dr. Victoria Shabardina ,
I think as your downstream process after DNA purification is the absorbance measurement, the presence of EDTA in your purified DNA sample might be affecting your absorbance results because EDTA is also absorbed in UV range around 258 nm. Therefore, like your downstream process is the measurement of absorbance you have to choose the best version of your buffer. The higher ratio of 260/230 might have resulted due to the presence of EDTA in the eluted sample. There is no doubt that the Tris-EDTA is the best for storing your DNA, but your absorbance reading is likely affected by the presence of EDTA
Sandhya Jayasekara
thank you again:) So, even if I blank against EDTA, EDTA will influence the measurement?
Dr Victoria Shabardina
EDTA remains as a chelated complex with Mg in DNA product. So this chelation, I suppose that might have an effect in your results.
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