@p-oberacker
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I just saw in this PDF link that I sent you that EDTA also causes peaks at 230 nm. However, the sample they show there is 100 mM, so 1000x concentrated compared to yours. Maybe try eluting with water and see how this looks. My money would still be on the carbs and phenolic though
Hi Mike,
No worries, glad if I can help!
Good to know! I will forward that to our webpage manager! That should not be the case.
Great! Glad the thorough washing did improve the quality of your samples. I agree, these ratios don’t sound good. However, as I personally never used the plant protocol myself, I’m not sure what to expect. Plants generally contain tons of carbohydrates and phenolics which might stick to the beads more aggressively, and both could lead to 230 peaks as well as far as I know. I’m not sure about ironIII though. Although it might cause such a peak, I doubt it would be carried over as it should be oxidized quite quickly. I found this nice PDF online showing different contamination graphs for NanoDrop. Does any of these look like your samples?
I will anyway forward this to the guys who developed this protocol and ask what their samples looked like in regards to purity. Maybe they can help shining some light on this matter.
Cheers,
PhilPS: How do you wash your beads? Do you fully dissolve them? If you pipette them up and down or even vortex them, you should be able to have them nicely dissolved, which definitely increases the efficiency of the wash step.
Hi Mike,
Great so at least the clumping issue is dissolved then (pun intended). Did you see any increase in yield, when adding more beads?
I don’t think the elution buffer is causing the GITC takeover. However, you could compare it by running a sample and elute with pure ddH2O, though, and see if that changes things. But as I said, I don’t think the buffer is the problem. How many times do you wash with ethanol? You could try to add two more washing steps. I usually do this with the RNA protocols and never experienced any takeover of GITC.
You mentioned that you’re following the animal tissue protocol #6.3. We also have a protocol specifically designed for plant tissue #6.4 which might suit your application better. However, the protocols are quite similar and only a few steps are different or swapped. As these two protocols were designed by our NZ team, I’m personally not too familiar with them. However, as only the initial steps are different and you get a good yield, I think it doesn’t make a big difference here. You might want to have a look, though.
Let me know if additional washing steps help you get rid of the GITC. If you still have troubles, I’ll forward your issue to our NZ team who are more familiar with TNA isolation from plant tissues.
Cheers,
PhilHi Mike,
indeed the absorbance at 220-230 nm is most likely GITC and yes, the two observations you made are probably connected. We usually see this clumping behaviour of beads when they are overloaded with nucleic acid. I posted an update addressing this on Researchgate (Project log Feb. 26: https://www.researchgate.net/project/Bio-On-Magnetic-Beads-BOMB). The flakes or clumps can obviously not be washed as properly as free beads leading to carryover of GITC and other things. I would recommend to either use less of the sample or more beads (maybe both). Let us know how it works for you.
Cheers,
PhilHi Sheila,
The COVID-19 protocol and the used buffers are based on our RNA isolation protocol #8.2, in which we purify RNA with silica-beads, so there shouldn’t be a problem. However, to my knowledge, we didn’t try this exact protocol with silica-beads yet, so no guarantee. But I don’t see why it wouldn’t work. Just give it a try 🙂 if you have any more questions, don’t hesitate to ask.
Cheers,
PhilWe usually use higher grade, e.g. N45. I assume N38 could also work, but expect the pelleting steps to consume more time. However, the racks in the link suggest prices as low as 18 $. In such a case one could scrape off the N38 magnets and easily replace them wit N45 or N48.
Well, if you check Google, there are dozent of suppliers, as e.g. Thermo: https://www.thermofisher.com/order/catalog/product/CS15000#/CS15000
You’ll just have to pay between 300 and 400 €/rack. Considering that there are even decent 3D printers in this price range, it would literally be more cost effective ato buy one and print the rack yourself.
Cheers
Phil@Alexander James Phillips, do these aggregates look a bit like flakes that form right after the addition of alcohol? I encountered this while testing bead capacity and uploaded the respective results on researchgate (https://www.researchgate.net/project/Bio-On-Magnetic-Beads-BOMB). Just check the update at the 26th of February. Simple reason would be overloaded beads.
Cheers
PhilHi David,
you’re completely right! Thanks for pointing out the discrepancy! The correct ratio is the one given in the main protocol, not in the modifications paragraph. We published a more thorough version containing the correct ratios with bio-protocol (https://bio-protocol.org/e3394) and I just formated the protocol on the homepage accordingly. Sorry for the mislead!
As Tim mentioned correctly, the 22.5 g wet mass are wet beads, they are not dried.
I hope your synthesis worked anyway.
Best regards
PhilHi Karina,
sorry for the late answer! Everything’s a bit crazy right now ^^
1) we never checked this, but I would assume it works with gram+ bacteria too. If not, one could think about digesting the cell wall before the lysis. We did something similar with yeast (#6.5).
2) Both bead-coatings should work with that protocol, although I personally prefer the silica-coated ones. I never tried these commercial beads, but don’t see why they wouldn’t work. However, other people had a few issues using commercial carboxyl-beads with our buffer systems. I would just give it a try or do a quick synthesis of BOMB-beads: https://bio-protocol.org/e3394
3) That depoends on the beads and what the supplier information says to do before using them. Our beads are simply suspended in water at a ratio of 1:1 wet mass volume to water volume.
4) I would again say, give it a try. We never tested this as far as I remember, but I don’t see why SDS shouldn’t work. Then again, I’m not a Chemist by training ^^
5) If you check in the original publication (https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000107) at Fig 2I our 260/280 ratios for gDNA are between 1.75 and 1.90, so yes, a bit higher is sometimes the case.
Hope it helps and sorry again for the delay. If you have any more questions, feel free to ask. I checked the notify button in this thread now.
Hey Isaac,
as we’re all based in Europe and New Zealand, we never checked for US suppliers, sorry. It is hard to say without trying though, if these alternative magnets work. However, the crucial point is the grade of the magnet, as one can always adjust the .stl file for smaller ones. I just fear that smaller magnets might be to far away from the well to attract the beads efficiently. But that’s what one would need to try out, maybe with a higher grade. I would definitely not try weaker magnets than the ones we used (N45) though.
Cheers