I have done 285 samples of human blood DNA extraction, and measured the DNA Concentration and A260/280 and A260/230 in a nano drop machine. This is done before PCR. Now the values of A260/230 are less than 1.5, most of them are 0. or 1. something.
Now After doing pcr and gel run, I want to do sanger sequencing, wil there arise any problem due to the purity value of A260/230???
The A260/280 ratio is a common metric used to assess DNA purity, with the ideal value being approximately 1.8 for pure DNA. The A260/230 ratio is also used for this purpose, and an ideal ratio is typically above 2.0. When the A260/230 ratio is less than ideal, it typically indicates the presence of contaminants such as carbohydrates, phenol, guanidine or other chemicals that absorb at 230 nm.
While a lower A260/230 ratio indicates potential contamination, it may not necessarily inhibit your ability to perform Sanger sequencing on the DNA samples. A lot depends on the nature and extent of the contamination.
However, impurities could potentially interfere with the PCR amplification step before sequencing or with the sequencing reaction itself, leading to lower-quality results or even failed reactions. Therefore, purifying the DNA further, if possible, is generally recommended before proceeding.
You could potentially use a DNA clean-up kit, or a phenol-chloroform extraction followed by ethanol precipitation, to help remove contaminants and improve the purity of your samples.
It's not uncommon to proceed with sequencing even when the A260/230 ratio is less than ideal, especially if other quality indicators (such as the A260/280 ratio and the quality of the PCR amplification) are good.
If you decide to proceed without further purification, I'd recommend sequencing a subset of your samples first to see if the quality is acceptable before sequencing all of them.
What extraction protocol did you use? Did you use EDTA in any step?
Would it be possible for you to repurify the samples?
I did this with some samples I extracted a few days ago with phenol-chloroform. The 260/230 ratio wasn't great so I tried repurifying and now my samples have much better purity indicators (260/230 went from 1.10 to 2.10).
An additional step of pellet washing with 70% ethanol may also help.
@Andrè Silva, I used Qiagen blood DNA extraction protocol, and yes I used EDTA for sample collection.
@Joshua Thank you so much for your valuable answer and it means a lot.
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