Hello,
I just started extracting DNA from some spiders, using 5-mm ball bearings to grind the specimens in a tissue mill. I had ordered the ball bearings on Amazon, and thought it was a good idea to sterilize them before use, so I soaked them in ~0.3% sodium hypochlorite for 30 minutes. Then I rinsed them with 100% ETOH three times and air dried them. Unfortunately they rusted during the bleaching, so that the bleach was completely brown when I poured it off. Still, I figured it would be all right to use them once (and discard after the first use) because they should at least be free of any DNA.
So I went ahead and used them to grind my specimens, then proceeded with extraction using the Qiagen Puregene Tissue kit. I did cell lysis overnight, roughly 20 hours at 55 degrees. When I removed the samples from the incubator, the lysate was clearly rusty (reddish in color with sediment in the bottom of the tube). At the end of the protocol, the DNA pellets were also quite red. I have done this protocol many times, and the difference in pellet color from the usual whitish/gray-brown was very apparent.
My question is, does anyone know if the rust will cause any harm to the DNA? I plan to use these extractions for metabarcoding, just amplifying short fragments of 400 bp or less. I will run a test PCR, but in the meantime I wondered if anyone else might have experience or knowledge about this particular issue.
Thanks in advance!
James Keane Bashkin and Anton Slyvka thank you for the follow-up info! Anton Slyvka it's interesting; the rust actually precipitated out on its own, so that the day after DNA extraction, I was left with very distinct rust pellets in the bottom of each tube. I ran a couple of test PCRs using the supernatant, and even very short fragments (100-150 bp) did not amplify for most samples. So it seems you are both right, and the damage is already done - severe DNA degradation probably took place during the cell lysis step. Sadly, those were irreplaceable samples - a good reminder that it's always better to test new consumables with some practice samples before going ahead with the real ones.
David Farringdon Spencer
I was sure we had bought stainless steel ball bearings, but I just discovered that "chrome steel" is what ended up getting ordered. So I guess that explains it. Not a mistake I will repeat!Thank you all again for your help. This has been a valuable learning experience for me!
Hi Susan,
yes, iron damages DNA pretty strongly, especially at high temperature. It is responsible for the generation of hydroxyl radicals which are the cause of the oxidative DNA damage.
Article Iron-induced oxidative DNA damage and its repair in primary ...
Article Metal-mediated DNA damage and cell death: Mechanisms, detect...
I would try to isolate DNA without the involvement of transition metals at all.
Best of luck!
Anton Slyvka is correct. The main problem with the iron will occur when any reducing agents are present because they help radical formation. However, most proteins are stored with reducing agents for enhanced lifetimes, so when you attempt to use this DNA in the presence of enzymes, it will be cleaved extensively, I expect.
Anton Slyvka and James Keane Bashkin thank you for your answers! Do you think I might be able to remove the rust via salt precipitation (basically a repeat of the DNA purification protocol)? I imagine that AMPure beads would also work, but unfortunately we don't have any in the lab at the moment.
You are very welcome. To answer this next question, I don't think it is practical. Removing even small amounts of metals from DNA can be tricky. For example, using Chelex 100 resin, which can be thought of as almost like supported EDTA because it has iminodiacetic acid groups, is not trivial. At high pH of 10-11, it can be used to remove Mg2+ and similar metals. However, it is not necessary to raise the pH to remove iron with Chelex 100 (pH of 4 would work fine, so don't buy the sodium form, buy the acid form). These pH differences have to do with the Lewis acidity of Fe(II) and Fe(III) ions, which is very high compared to that of Mg(II).
The problems are several-fold. You undoubtedly have Fe(III) in your sample, and that is typically insoluble except at very acidic pH, except that the DNA is probably solubilizing it by binding, and that binding isn't so easily reversed.
Another problem is that iron bound to Chelex can still damage DNA, even when present in very small amounts, so it is important not to leave the DNA on the Chelex overnight, for example, but rather for a few hours (I don't have an exact protocol in mind, it has been too long since I did this last). With the vast rather than trace amounts of iron that you have mixed with DNA, I think you will be much happier starting over than trying to rescue this batch of DNA and ending up destroying it in the process of additional purification.
Chelex works to remove relatively small amounts of metal ions, not massive amounts of them. Really, even if you removed 90% of the iron, which is optimistic, the remaining metal would be more than enough to damage your DNA extensively. Sorry I can't be more optimistic, but maybe someone else will have better ideas.
Best wishes, Jim
The only strategy I would consider is pelleting the insoluble rust and then passing the supernatant through mini size-exclusion collumns.
(for instance: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/11814427001bul.pdf )
I would repeat it 2x or even 3x and this should (but not necessarily) remove iron traces.
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