I start with impure PCR products of >500ng/ul, 260/280=1, but peaks present at 260, and end up with peaks near 230, about 10ng/ul concentrations of 260/280=1.5 or so, which I assume is not DNA since there is no peak at 260. Is this due to contamination by guanidinium salts from the buffers? Or ethanol? What can I do? Gel electrophoresis with a 2% agarose gel for 25 min at 70V showed DNA of the desired sizes, though not necessarily large amounts, and after purification, there was pretty much nothing.
Hi Melissa,
Column-based methods shoud be work well for DNA purification tasks. But sometimes from many reasons (high pH (7.51.5), respectively.
Yes you are right in the terms of contamination. Ethanol (Wash Buffer PE (QIAGEN) and Monarch DNA Wash Buffer (NEB) in the your kits contain it), EDTA, carbohydrates, guanidine HCL (used for DNA isolation) and phenol has absorbance maximum at 230 nm. But in your kit the most probable contaminant is the ethanol. Anyway A260/A230 ratio should be ~2.0-2.2.
You can purify your DNA with sodium-acetate-propanol precipitation. I have a protocol if you need. Or you can add the DNA sample onto a fresh column and follow the protocol from the wash step again. With both methods you will lost yield, but your DNA will be more pure.
Sodium-acetate-propanol precipitation method's
pros:
- more cheaper than any other column based method
cons:
- problematic to do in high throughput compare to column-based methods, but if you haven't got to much sample (e.g. below 12) it's a good approach to purificate your DNA.
Bests,
Tamas
Looks like guanidium band to me. Good news is that little blip at 260 is probably real DNA. Bad new is there isn't much of it. If the pcr band looks bright on gel and there wasn't much off-target amplification, I'd re-run the PCR using the gel-cleaned stuff you've got as template, then use a PCR cleanup as others have suggested. Good luck!
Hi Melissa,
With that Qiagen kit I always get a huge absorbance band at 230 nm that hides my DNA too, unless I have heroic amounts of DNA. If you run an aliquot on an agarose gel, you will see that the DNA is there (at least, it always has been for me!).
What do you want to use the PCR product for? I have successfully used DNA purified using this kit for direct Sanger sequencing of the product and for ligations without requiring further clean-up.
Philip Kitchen I need to use that PCR product in a subsequent PCR reaction for SOE PCR, and the protocols that I've read say that I should ideally use a 260/280 ratio of >1.7
260/280 would usually tell you about protein contamination, but if you have some other contaminant with broad absorbance spectrum, a low 260/280 is not necessarily fatal for your experiment.
I would run an aliquot of your product on an agarose gel to convince yourself that you have purified a decent amount of DNA (you could also use the intensity of your band relative to the closest marker band to estimate DNA concentration), and if so then just try the SOE PCR - it may be that the contaminant is not an issue.
If the SOE PCR fails - then think about re-purifying your DNA using NaAc/columns/Ampure etc.
Philip Kitchen The intensity of my PCR product on the gel was relatively faint, so I may have to try to reoptimize my PCR again. Thank you for the advice. Which method have you found is the best for purifying your DNA post-PCR?
I mainly purify PCR products to either clone into vectors or Sanger sequence. For these applications, the contamination from the Qiagen gel extraction kit has never been a problem.
For purifying DNA in NGS workflows, we use Ampure magnetic beads, so you could try that if the contamination from the gel extraction kit inhibits your second PCR, but I suspect it won't be an issue.
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