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…Read more

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…Read more

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:…Read more

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…Read more

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 y…Read more

Thanks! That is actually important to know!

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.…Read more

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…Read more

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…Read more

Cheers
Phil

getting RNA from yeast shouldn’t be a problem. With the original paper we aslo published a protocol to isolate TNA from Saccharomyces (#6.5). We basically just break up the cell wall with a lyticase digest and go on like in any other BOMB TNA extraction. That also means, that allows you to basically switch to one of our RNA protocols…Read more

1. As Tim mentioned correctly, clumping of the beads is in my experience a sign that the beads reached their capacity limit. Is it only happening during the washing steps, or already before? I recently uploaded an update on bead capacity and clumping behaviour (26.02.20) in the project log of the BOMB project on Researchgate. Maybe this…

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we just released a protocol addressing exactly this!

https://bomb.bio/wp-content/uploads/2020/04/SARS-CoV-2-RNA-purification-from-nasal-swabs_BOMBv2.pdf

Cheers,
Phil

In the step before the addition of GITC you do a proteinase K and RNase A digest (depending on what you want to extract, TNA or RNA). GITC in general denatures proteins. While proteinase K activity can actually be enhanced at lower molarities, I don’t know how it will effect the enzyme a the concentrations used in the protocol (4-6 M).…Read more

I talked to our synthesis specialist about your issue and he mentioned that the main issue for clustering is a high MNP concentration. So if you do the whole procedure diluted, either by using less MNP wet mass or do it in higher volumes, you should be fine.

Let me know if it worked! 🙂

Cheers
Phil

both protocols will work fine. The main difference is that we add the TEOS later in V1.3. This is to ensure the ammonia is equally distributed and the reaction conditions are perfect before starting it with the addition of TEOS. We see a better yield in coated beads, but if you synthesized with V1.2 that’s fine too. However, for the…Read more

we’ve actually published a follow-up paper to the original that focuses on the bead synthesis on bio-protocols (https://bio-protocol.org/e3394) which contains exactly that. In there you’ll find not only a list with catalog numbers of all used labware but also a picture of the actual set-up (Fig 1A). The bowl for the water bath (no…Read more

as I said on Twitter, it’s a great design and it will be a perfect addition to the 3D printing section! Thanks a lot for the contribution!

Cheers
Phil

there is a protocol for plant tissue TNA extraction available (#6.4). The protocol also contains a gDNA version, as you simply do an RNase digest after lysis.

Cheers
Phil

No, this is actually not supposed to happen and I can’t say we ever encountered this issue. Besides the beads, ammonia, and TEOS, the solution is basically 2.5 litres of 80% EtOH. Such a solution should not be completely evaporated after 4 h at 80 °C. Maybe the temperature was set to high? However, if you add a reflux funnel on top of…Read more

I can only support trying to find individual do-it-yourself methods as that’s how you really understand what you’re doing 😀 otherwise you’ll become a kit-scientist 😉

I would indeed recommend the environmental TNA protocol (#6.7) as a reference. Dissolve your soil sample in e.g. water or TRIS-buffer (in the protocol we went with 50 ml…Read more

as Tim already said, the key is to add the iron solution in a dropwise manner to the NaOH, while constantly stirring the latter. For this, we use a round-bottom flask with the NaOH in it and place it on a magnetic stirrer. We then put a dripping funnel on top in which we add the iron solution. You can find a thorough description of…Read more

@svitak these might be interesting for you! Look for #5.3 and #5.4 (https://bomb.bio/protocols/)

First and foremost, thanks a lot! We’d be happy to include your protocol as a community contribution to the BOMB protocol collection. As there have been multiple people now who had troubles adapting the BOMB protocols to Sera-Mag beads in the last weeks, your protocol would be of great benefit for them. I’ll forward your offer to…Read more

In general, the AMPure XP beads should work as well, with our buffer system, however, please keep in mind that they have a different structural composition than the BOMB beads. While the AMPure XP beads have a polystyrene core covered in a magnetite and a carboxyl layer, BOMB beads have a ferrite core and a carboxyl surface. Therefore,…Read more

Sorry for the late response! We’re just moving our lab so there’s quite some work to do away from the laptop ^^

Firstly, thanks a lot! We’re glad you like the protocols 🙂

To your first question: The Sera-Mag Beads are built a little bit differently than our beads. While Sera-Mag have a polystyrene core coated with one or even two m…Read more

Thanks a lot for the thorough feedback! Findings like these are excactly what we need!

Cheers
Phil

Mixing the beads with the ethanol before adding the supernatant is perfectly fine. In this step it’s only critical to have the ethanol in there, as it drastically increases your yield.

Yes, it’s expected that the beads will take up some volume, especially if they were dried thoroughly.

I hope you were able to isolate some good yields…Read more

We have indeed not done this for RNA as small as microRNAs. However, the binding of DNA as well as for RNA to the silica-beads depends on the salt concentration during the binding step. For DNA we recently developed a protocol for nucleotide removal (#4.4), where we capture DNA oligos with a length of about 25…Read more

this is the main discussion space for BOMB protocol #4.4 BOMB Nucleotide removal using silica-coated MNPs
Here you can post all your questions and feedback!

this is the main discussion space for BOMB protocol #4.3 BOMB Agarose extraction
Here you can post all your questions and feedback!

this is the main discussion space for BOMB protocol #5.2 plasmid DNA extraction from E. coli using MNPs for capturing cellular debris, instead of the common centrifugation step.
Here you can post all your questions and feedback!

Thanks for the input! We’ll definitely add example models to the respective protocol.

To your question: for the setup and batch size described in protocol #1.1 we usually use a reflux funnel (250 ml, NS29/32) from Buhler and a round bottom flask (1000 ml, NS 29/32, PN: 2172154) from Schott Duran. For the water bath we use common h…Read more

this is the main discussion space for BOMB protocol #4.3 BOMB Gel extraction
Here you can post all your questions and feedback!

this is the main discussion space for BOMB protocol #4.2 BOMB Clean-up and size exclusion using carboxyl-coated MNPs
Here you can post all your questions and feedback!