Hi all,
I'm trying to clean up some DNA for genomic sequencing (Nanopore and Illumina), but i'm still struggling with the purity of my DNA.
My 260/280 ration is ok, that is not an issue, however my 260/230 is always lower than 1.6.
Firstly I thought it could be contaminants carryover from my extract, but I just ran some tests on Lambda phage DNA and got the same problem.
I got some lambda DNA and run some tests with addition of different volumes of my homemade Ampure beads (https://openwetware.org/wiki/SPRI_bead_mix).
I tried 1:1, 1:2 and 1:9 and my 230 ratios were again lower than 1.5.
Is it possible the beads are carrying some PEG? If so it wont be a problem for the libraries preps since the ligation buffers uses peg as well, right?
Can you see other source of 230nm contamination?
Thank you in advance,
Caio
Dear Caio...
Generally, Low or high value of 260/230 may refer to a problem with the use sample or with extraction procedure. You have to follow the next
1. A low A 260/A230 ratio may be the result of:
• Carbohydrate carryover (often a problem with plants).
• Residual phenol from nucleic acid extraction.
• Residual guanidine (often used in column based kits).
• Glycogen used for precipitation.
2. 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 a similar ionic strength as the sample solution.
Example: Using water for the Blank measurement for samples dissolved in TE may result in low 260/230 ratios.
(Reference: William W. Wilfinger, Karol Mackey, and Piotr Chomczynski,
Effect of pH and Ionic Strength on the Spectrophotometric
Assessment of Nucleic Acid Purity: BioTechniques 22:474-481
(March 1997)) Cited by Tewdros Endalew.
Thanks for you answer Ayoob,
I thought it could be that, as I was told that DNA can make covalent bonds with EDTA if you have remaining EDTA with your DNA during the ethanol precipitation.
I also was told that Ampure beads co-purify some mysterious 230nm contaminant from fungal DNA (empirically observed).
Knowing that, I tested the Ampure clean-up from a commercial Lambda Phage DNA.
The starting 260/230 ratio was 1.84 and after my beads clean-up, the ratio dropped to 1.45.
I am testing the same homemade ampure recipe but without the EDTA to check if this may be the problem.
The higher than expected absorbance reading at 230 nm may not be a problem. As pointed out in the previous answer, it can come from a variety of sources.
Are you taking the DNA directly into the library prep or are you amplifying it first? If you can show that there is no inhibition of PCR or ligation reactions, you could just use your DNA even if the OD260/260 ratio is lower than you would expect it.
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