https://bomb.bio/protocols/   –> Nucleic Acids –> #7.1 DNA extraction.

Cheers

I dare say that what you posted in itself is not a functional DNA extraction method.

Unless you are also adding some sort of solvent or additional salt to your wash buffer, there is no reason why your wash buffer wouldn’t wash away the DNA you are trying to capture.

If, the issue isnt in your wash step then i would assume the issue might…Read more

Seems like all I read was that an alternative to the GITC bufffer is needed, without actually looking at what it is used for.

Cheers

Tim

Have a look at this link, I would say it sums up pretty much what is achievable using  SPRI :

https://ls.beckmancoulter.co.jp/files/appli_note/Gel_Free_Using_SPRIselect.pdf

(I am in no way affiliated with this…Read more

The Urea+LiCl  lysis…Read more

I too have experienced significant issues when trying to isolate DNA from overly contaminant rich sources, massive amounts of white precipitate etc.

My guess was that this has to be some sort of water soluble proteins which will get bound to mag-beads once precipitated.  Could you try incorporating a proteinase K step?

Tim

I am stocked to hear that you like our protocol!
I presume that you have thought through whether RNA contamination actually poses an issue.
Because unless you are doing super sensitive quantification, transfections or steps involving reverse transcriptase, I never really encounter any issues whatsoever with residual RNA. But, I am mostly …Read more

In a way I hope you already solved the challenge and I am therefore too late.

But maybe not so I figure I may as well share my thoughts.
I do not know the exact composition of Qiagen N3 but since it is a silica binding buffer I would assume several things that may not be ideal for your purpose.

• DNA (Including Plasmids) is already…

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yes indeed.

Since I am more or less the only person (aside from visitors/participants) keeping this forum alive (and mostly spam free) I had to restrict the number of allowed links per post as that is what bots absolutely love to post. Sorry about that.

As to the protocol. I just had a read through and I would mostly concur. I did not…Read more

The process is often referred to as “nitrogen stripping”,  on the fly I found this useful publication on the matter.

https://www.sciencedirect.com/science/article/pii/S1388248119300980#f0005

How long and how much depend on flow, bubble size etc. As far as I am aware this is usually done for an excessively long time, so that oxygen…Read more

since the binding process of both carboxyl and silica beads is predominantly ionic in nature I agree that Beads aren’t nearly as discriminating as one would hope. However the trick is in the binding solution/buffer, if you use too much salt or solvent you will indeed purify proteins along with DNA/RNA.

From my limited experience with…Read more

This seems like a promising source for information:
https://link.springer.com/article/10.1186/s12885-019-6363-0

Best of luck!

https://www.sciencedirect.com/science/article/pii/S0141813019305537#f0065

Cheers

Tim

Just so you do not feel like the bomb team is keeping a protocol in secret for making cellulose beads.

Unfortunately this is not the case. I have personally not even been aware that they exist/are being used until now.

Please, should you find something, let us know, we are happy to host good bead protocols (CC).

Cheers

Tim M

Welcome to the forum.

First of all the 300 kg pull-force sounds even more scary than it is.
The magnetic field flux density follows an inverse square law and thus a small magnet,even one with 300kg pull force has very little effect on anything further away than a couple of centimetres.

Unfortunately this is also the reason why we…Read more

Way, way late so I truly hope you have found a solution by now.

If not, how about you dilute your PCR product prior to adding the binding buffer?
This way the binding solution buffer capacity should overcome the PCR mix buffer capacity.

e.g. rather than using 10ul PCR product and 15ul of Binding buffer (1.5 ratio)

you could use

10 ul…Read more

I am intrigued but admittedly have never even come across this as an option.

However, the streptavidin/biotin hydrogen bonding can be achieved in absence of free ions whereas normal silica or carbox bonding relies on a highly ionic surrounding. Hence you would probably have to apply rather significant charges to…Read more

Unfortunately the answer to all your questions is:

Has to be tested empirically.

It is not possible to estimate/calculate the quantity of Beads needed to achieve best results. This can only be determined empirically. Size dispersity has too much of an effect on bulk density to know the number of beads per volume. If you think of a big box…Read more

I have myself encountered this problem and do not fully understand why it sometimes occurs but not always and not for everyone. But it is most certainly the result of Oxygen/oxidation. Aside from the usual recommendations which you seem to have already adhered to I can at this point only recommend to find the means to perform your synthesis…Read more

I have recently performed a vast amount of empirical trials on silica binding conditions and I can tell you with complete confidence that you will not be able to fully theorize the best conditions.
I tried different batches of homemade Beads, different Commercial beads, different lysis/binding buffers, different alcohols and percentages and…Read more

For information on Ethanol/Iso. I recommend this:

DNA Precipitation: Ethanol vs. Isopropanol

The gist is that both work fine but you only need about 35% Iso whereas you need roughly 75% ethanol. Since you already have the lysis buffer and bead storage buffer in your tube you would have to add an inconvenient…Read more

Thank you for your interest.

This can be attributed to the individual protocols not having been created at exactly the same time and researcher, resulting in minor adjustments between them.

As to what is the better procedure.

The EtOH wash step is mostly aimed at washing off residual salts. Predominantly this is achieved through the 20%…Read more

I will ask the person who contributed this particular protocol whether she has any idea as to how this can be solved.

Cheers

Tim

Welcome and thanks for the flowers.

Unfortunately I cannot give you a clear answer. This seems to bee a rather common question on the interweb, sadly with little to no answers. I would suggest trying Fuchsin (based on this article https://jb.asm.org/content/jb/52/1/99.full.pdf).

Colour wise it is strikingly close to the one in probrietary…Read more

yes the Beads will settle within a few minutes or less, depending on size and magnetic interaction. We strongly recommend to always shake up the beads before you take an aliquot. Especially once the beads are diluted to working strength for your respective protocol, make sure you aspirate with a pipette or shake before you take up the…Read more

Thanks Thomas you are absolutely correct.

I changed it and uploaded an updated version where I corrected the product number.
As all I did was add a single digit I figured it would be ok to just upload it under the same version number (V1.0).

The change should take affect as soon as all caches are refreshed.

Cheers
Tim

Cheers
Tim M

Until a moment ago the link for BOMB protocol #6.4 TNA extraction from plants was erroneously leading to this thread which is meant to discuss #6.3.

All plant related posts that were posted here before have been moved to the correct thread and the link has been corrected.

Best Regards
Tim M

Some further info.

I fully agree. Use water for storage and TE for working solution. As bead synthesis requires harsh conditions it is advisable to re-suspend them in a buffering medium to guarantee that any remnant hydroxide anions (silica beads) or hydrogen cations (carbox. beads) are neutralized and wont interfere during consequent…Read more

While I am a part of the original BOMB team and have been using many of the protocols extensively, as well as some of the beautiful beads that Tomek made, there is an aspect that I feel like I should be able to answer but cant.

If one could freely choose, what would be the ideal Magnetite Core Particle size for consequent silica…Read more

to start off very cheesy. You are not alone.

With an increasing number of people facing issues trying the BOMB 1.1 MNP synthesis, I have spent the last couple of weeks looking into what could be the source of these issues and sadly I have not figured it out completely. But I got a couple of time-saving remarks for anyone who is…Read more

While I am not Phil I’ll try to answer your query.

I presume you are referring to the “BOMB-Community 2pt rack” by Malgorzata Cebrat ?

I have just had a look and the two individual components are both available in their respective files.

Please have another look and download them both by simply clicking on the pt1-stl and the…Read more

My sincere apology for completely having missed your question. Considering how long ago you wrote it, I hope you have successfully silica coated MNPs in the meantime.

Unfortunately I cannot answer your question concerning the odd ratio difference. To be honest looking at the protocol I am as befuddled by this as you are. However, I…Read more

Thank you for this valuable feedback. We do not have the file on github but I will see to it that we upload an edited version asap. Do you happen to have any information on a time-frame within which this became an issue for you? While I have premixed isoprop and beads without any issues I have personally never done so more than a few…Read more

Happy Easter everyone. I hope things are going OK.
@Bradley/Emily:

I guess you will ultimately choose one or the other Protocol. Have you made this decision yet?

Cheers

Tim M

I don’t think anyone from BOMB is currently planning on doing so and I do not know whether Canterbury health has the capacity to go through the rigamarole of scientific publishing while providing the COVID-19  testing service at the same time.  I will raise this issue with my colleagues asap.

Cheers

not at all.

While I have not used the protocol myself I know that the other part of our team who came up with it recommend to place a magnetic stir bar in both the water bath as well as the round bottom flask in order to avoid heat accumulation.

Also, I fully agree that the drawing in the protocol is a bit unclear in that regard.

Cheers

Tim M

Three things.

First I have actually tried using the BOMB 2x GITC Buffer without Antifoam. I did not test for actual efficiency as this was just a rushed little experiment were the focus was on testing a new enzyme down the line rather than the extraction itself. The reason I left out the anti-foam was simply that I was in a hurry, had to…Read more

Cheers

Tim M

All replies concerning adaptation of this protocol for SARS-CoV-2 RNA extraction have been moved to

Adjusting BOMB protocol # 8.2 for SARS-CoV-2 extraction

Adjusting BOMB protocol #8.1 for SARS-CoV-2 extraction

Unfortunately I am not as savvy with the molecular processes behind what is happening as other members of BOMB team who will hopefully chime in.

1. Using the 8.2 protocol I have encountered clumping of beads in samples I knew were a bit too concentrated (contained too much tissue). However this never led to any problems down the…Read more