[admin]

I hope you have had some success? I am unaware of any wash step that does not rely on DNA remaining precipitated (= alcohol present) to stay bound. I would really recommend you just try something like the straight forward BOMB protocol #7.1 :

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

Cheers

[admin]

Ahoy,

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 be with plasma being very high in proteins and quite low in DNA. It could be that you are facing issues with proteins overwhelming the binding capacity of your beads. Silica beads are great at binding DNA but also not very specific, they will bind proteins as long as the right charge difference is given. I take it you are adding isopropanol to allow for binding? If so, how much? Going as low as 35% isopropanol (final c. including binding buffer/beads in solution/sample) could possibly help by reducing protein precipitation.

Lastly, are you proteinase K treating your sample? If not then I would strongly recommend this as a troubleshooting step.

Cheers,

Tim M

[admin]

Welcome back Sebastian and thank you for picking up on what I so blatantly overlooked.

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

[admin]

I personally have always had to resort to agarose gel separation and extraction when trying to selectively purify at this small size.

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 company, have never purchased any of their products and can neither advocate for or against them. However, I do like their graphs.)

Cheers

Tim M

• This reply was modified 5 months, 2 weeks ago by admin.

[admin]

Ahoy,
With the forum having grown rather quiet over the last year I suspect Sebastian might not see your request. And while I am not willing to dox him I dare to say that you might be able to find his e-mail via google.
Anyhow I can also suggest the following as a binding/extraction buffer which has worked very well for me:

The Urea+LiCl  lysis and binding buffer recipe for 50 mL :

6M Urea (18g Urea)
3M LiCl  (6.3 g LiCl)
500 mM TrisHCl (2.5 mL of 1M stock, pH 8)
30 mM EDTA (2 mL of 0.5M stock)
0.5% Tween20 (250 uL)

I used this to replace the standard BOMB GITC buffer 1:1.

This buffer is a lot cheaper, less toxic and performed about as well as the GITC variant. There are two main downsides to this buffer compared to GITC, it does not keep as well, after about a week at room temperature I would always find it to start performing somewhat worse and at some point it would develop a shiny film on the surface and it has a tendency to precipitate when room temperatures are cool. I believe the short shelf live to be some form of Urea deterioration and not microbial contamination but I never tested it as the tiny extractions I perform require so little of the GITC buffer with its nearly infinite shelf life that it was just not worth switching to this alternative permanently.

In case you feel like this will be difficult to acquire, ask someone in a lab which has been doing RNA work for a while, there is a good chance the have high concentration LiCl solutions, as for Urea, it is even cheaper than LiCl.

Cheers

Tim M

 

 

[admin]

Ahoy,

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

[admin]

Ahoy,

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  running PCRs or similarly robust techniques.

I am not sure whether RNAse A has a tolerance for Lyticase and GITC but I would not risk it.

Unfortunately the save option would be to finish the protocol transfer your elution to a tube loaded with RNAse A + Buffer –> Incubate  (+ maybe deactivate) and then perform something like a PEG (SPRI \ Ampure) purification or if you want to go DIY all the way BOMB protocol 4.1., obviously you can also ethanol precipitate etc at this point.

Cheers

Tim M

 

[admin]

Ahoy,

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 precipitated -> spinning may lead to some loss.
• The precipitated DNA in N3 will be charged and therefore sticky -> changing tube leads to loss.
• Adding ethanol at this point might lead to salting out and general precipitation of unwanted contaminants.

I do not understand why you would presume that you would be selectively binding plasmids in the procedure you describe.
All in all I think what you propose wont work but maybe you have already tried it and can prove me wrong?

However, could you consider simply using Exonuclease 5 (V)?
From personal experience ExoV is amazingly good at chewing up anything non circular.

Cheers

Tim M

[admin]

Ahoy,

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 the cited publication about the cellulose solution but other than that yes it seems like “this protocol really bind cellulose permanently by just dripping it in alcohol”.
However, the resulting beads are huge. I cannot imagine how you would successfully employ 1 to 2 mm “balls” for your purpose.

Since, as I mentioned before, I have got zero experience or knowledge concerning cellulose beads I am inclined to recommend  something I am far more familiar with.
Try alternative binding buffers.
I am inclined to say the buffer is at least as important as the binding surface.
Also did you give the beads from the link I posted a try?
What do you mean by better results? Purity? Yield? Ease of use?
Cheers

Tim M

[admin]

Ahoy,

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 presence is likely to be low or otherwise in the presence of an oxygen probe so that one actually knows when enough is enough.

However for your purpose I would simply let the N2 bubble through the water column for 30 minutes. I think the even more beneficial effect you would get from having nitrogen rather than air in the round bottom flask during the synthesis process.

For this I would use a three neck flask with one neck for the drip feed one neck for the N2 inflow and one neck partially but not fully blocked (idially with a valve) so that N2 accumulates but does not build up pressure within the system.

We have had a lot of scientists come back to us that did not remove oxygen, yet encountered no issues whereas others seem to encounter oxidation problems no matter what. Dissolved oxygen levels vary hugely depending on parameters surch as the water source, the surrounding pressure (altitude) and most of all temperature. Unless you have the means to really control and measure all aspects of this semi precise wet chemistry approach I would suggest empirical testing and simply trying it as best as is feasible.

The longer you bubble N2 through the system the less O2..

The hotter the liquid the less O2 ..

The less O2 is present during the vigorous stirring the less will diffuse into the liquid ..

But when is it enough? Unfortunately I have no idea.

And one more thing, maybe even more important than going over board on the de-oxygenation front is the quality of your iron sources. If the F2 is already oxidised further (and isn’t actually F2 any more) then you are in a way actively adding the Oxygen you are trying to avoid. The FeCl2 should be white to green (depending on H2O amount) with little to no hint of orange.
I hope this has helped, even just a little.

Cheers

Tim M

 

 

[admin]

Ahoy,

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 restriction enzymes you will never really get rid of them without inactivation. And, with inactivation they may still be present but I would not know.

If you are asking because you want to know whether it is ok to have beads present during PCR or Digestion.

We have some commercial carbox beads here that clearly say that they inhibit PCR to a certain extend, whether through binding or degradation products I do not know.

I have personally run PCRs with DIY (bomb) silica beads sitting in the tube and it did not seem to interfere.

I would however never do that for difficult/low input samples or something as sensitive as a qPCR.

To sum it up, I think you will have to empirically test whether it poses an issue in your specific application.
Cheers

Tim M

 

[admin]

Not on our end.

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

 

Best of luck!

[admin]

I do not know whether these are suitable for binding Nucleotides but maybe it gets you a little further:

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

 

Cheers

Tim

[admin]

Ahoy,

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

[admin]

Well yes, I cannot say I did not end up pinching my fingers a few times.
Just remember, it is only once the magnets get really close to each other that the force becomes huge. Hence you usually don’t smash fingers but are more likely to, well .. pinch skin.
So maybe just wear some leather gloves while you assemble whatever you end up creating.

Concerning a  suitable web shop, unfortunately I cannot help you there, I have not been in Europe in years and I just spent some time searching and could  not find the exact Magnets we specified available at any European shop either.

I did however come across this:

Here are my first designs of custom 3d-printed labware constructed with @adskFusion360 software and printed with @formlabs Form 2, at @PlantEvolution lab! #3Dprinting

(1) A magnetic rack for 96-well plates (23 Euro material cost): pic.twitter.com/TPlHSNASRt

— Uli Lutz (@ulrich_lutz) November 27, 2017

If he is willing to share the 3D files with you, those magnets look like they should be purchasable.

Best of luck

Tim M

[Author]

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