[TimH]

Hi Bernardo – sorry for the slow reply – we haven’t done a huge amount of testing using the hydrophilic beads. Did you get any further with this? We have done plenty of NGS (Illumina mostly) using GITC and the hydrophobic Speedbeads  – it doesn’t appear to cause us issues….could you clarify if its an issue for you? As above, we usually only get clumping when a lot of input tissue/cells is used and high-molecular weight DNA is used – but others (e.g. Sebastian above) have had clumping issues when extracting plasmids.

Cheers, Tim H

[TimH]

Hi Emily,

Blood does not really work that well with the 2:3:4 ratio, as there is a lot of liquid in there and (for humans/mammals) not many nucleated cells. You are probably better off going with a 2-step ’tissue’ protocol (#6.3), where you first protK treat in TNES, and then add in 1.5X GITC. From this point on it is basically 2:3:4.

Best wishes, Tim H

 

[TimH]

Hi Miriam – our group generally use deep well plates (1-2 mL) so when mixing using 1000 rpm on, for example, an eppendorf thermomixer, you won’t have to use seals at all.

Best wishes,

Tim

 

 

[TimH]

Hi Mike,

Sorry we are slow to the conversation, however, I think Phil has covered off the points I would say are important. Paramount is more stringent washing of beads – I strongly suspect the 230 peak is GITC contamination; especially as you point out you see it even when there is no plant tissue at all (so this is a general bead issue, not just plant protocol). I would compare it to commercial gel extraction kits, which always produce a peak at 230, presumably because of GITC carryover in the column. I think the pertinent question is probably, what level of GITC carryover are you comfortable with? A little bit of carry over is not an issue (as in gel extraction), but too much and it can inhibit downstream enzymatic reactions. We have always managed to find a balance we are happy with.

Cheers,

TimH

 

[TimH]

Hi Sheila – I would use TE – as Phil mentioned in your other post silica beads should work very similar to the carboylated ones we got the initial protocol optimised on. So keeping everything the same would be the best starting point. Best wishes, TimH

[TimH]

You can find this information in protocol #6.1 on our main page, or find below in the poorly formatted copy/paste from this page. But chemicals of good quality sourced elsewhere will very likely be fine. (note, you have details of the beads already)

Ethanol (C2H6O, 99.9%) Honeywell/ Riedel-de Haёn 34963

Guanidine isothiocyanate (GITC, C2H6N4S) Roth Chemicals 2628.4

Tris(hydroxymethyl)- aminomethane (Tris, C4H11NO3) Roth Chemicals AE15.3

N-Lauroylsarcosine sodium salt (Sarkosyl, C15H29NO3Na) Sigma (Merck) L9150- 50G 2

Antifoam 204 Sigma (Merck) A8311- 50ML

Ethylenediamine tetraacetic acid disodium salt dihydrate (EDTA (C10H14N2Na2O8 · 2 H2O) Roth Chemicals

Isopropanol (C3H8O) Acros Organic 18413002 5

[TimH]

Hi OMC Healthcare – this bead works very well in our hands, but other beads are also likely to work well (including self-fabricated beads, as in our protocols). We’ve not detected any sign of PCR inhibitor – remember that the beads are washed in the preparation steps, and then go through purification involving more washes, before being resuspended in MQH20. So I’m not sure where this inhibition would be coming from but happy to hear more details if the customer service rep will provide it.

Best wishes,

TimH

[TimH]

Hi Emily – details for the beads used in the COVID protocol can be found at the bottom of the protocol page (GE Healthcare Sera-Mag Magnetic SpeedBeads Carboxylate-Modified Dia.: 1 μm; GEHE451521 05050250) along with details on their preparation.

We’ve not tested BOMB silica beads for SARS-CoV-2 yet, however, this will happen soon. In the meantime, you might be interested in the direct comparisons between Speed Beads and BOMB silica beads we previously did for DNA extraction – there was no difference (see Figure S4 of Oberacker et al., 2019), but both were generally better than Phenol-Chloroform.

Best wishes,

TimH

 

[TimH]

Hi Alejandro – best of luck with your efforts. The anti-foam is an optional component as far as I am aware – it is the GITC and detergent that will lyse cells (and virus). So if you don’t have it, then you might want to still try without it.

We have not tried the V&P tool as in the video. I would think it is fine in principle, however, to avoid contamination you would need a new microplate and wash buffer for every plate. So might not actually save much on plastic ware. Robotic automation, including OpenTrons will work (likely more details/protocols will emerge around this soon); however, multichannel pipettes are also fast and effective if you don’t have one. The most time consuming aspect is usually getting the lysate from the swab in individual tubes into a 96-well format.

Best wishes,

TimH

[TimH]

Hi Bradley,

Many thanks for the message and sharing your protocol. Do you have a way to show/test inactivation of the virus? Commercial AVL is ineffective at inactivation of Ebola (https://journals.sagepub.com/doi/pdf/10.1177/1535676017703383), so please take this into consideration if/when you start working with actual virus. Be safe. You could consider trying a GITC buffer with some detergent as this may improve inactivation (e.g. our GITC lysis buffer from the ‘TNA’ protocol #6.1 as below):

GITC 4 M 23.64 g
Tris HCl pH 7.6-8.0 50 mM 2.5 ml of 1 M stock
Sarkosyl 2% 1 g
EDTA 20 mM 2 ml of 0.5 M stock
Antifoam (optional) 0.1 % 50 μl
adjust pH with HCl to 7.6-8.0 and adjust the volume with water to 50 ml

This protocol (TNA #6.1) also has also been used routinely with SpeedBeads as you propose, however, we prepare them in only TE (i.e. no salt), and at 1:50 dilution from the original stock, following 2x TE washes to get rid of sodium azide and other components of the storage medium.

 

Best wishes,

Tim H

 

[TimH]

Hi Alexander,

We are working with several facilities involved in virus testing to optimise modified BOMB protocols for RNA extraction that are both effective and safe. We will update on this as soon as we can, but we would like to underline our commitment to open science – precisely what is needed now when considering failing supply-chain and lives at stake. Protocols and results will be released as soon as they can be.

Best wishes,

TimH

[TimH]

Hi Jon – to prepare those beads for gDNA, RNA and TNA extraction take 1 mL of thoroughly resuspended bead mixture and place on a magnetic. Once beads have settled, remove the storage buffer (it contains sodium azide) and resuspend in 1 mL of TE buffer off the magnetic. Repeat process at least twice more and then resuspend in TE. We do this into 50 mL of TE – seems to be a good concentration of beads, however this could likely be altered without making much difference.

Cheers, Tim

[TimH]

Hi Sarah/Phil – just a quick message to confirm that for the TNA from tissues purification using Sera-Mag or BOMB beads; exactly the same protocol was used. As Phil mentioned, there was slightly higher yield for most tissues using BOMB beads…

Cheers,

Tim

[TimH]

.

[TimH]

Hi Sebastian,

Many thanks for the detailed feedback. I have tried to split the two main challenges you raise so it is easier for others to follow. However, due to the common issue of clumping, I suspect the answer for both is the same (you need to more beads for capture, or less starting material):

PLASMID EXTRACTION

You are right, most of the protocols are optimised for the lab-made ‘BOMB’ silica beads, so how well the carboxylate “Speed Beads” work in various situations has not always been tested or optimised. Plasmid extraction is one of those.

Nevertheless, I have done a lot of DNA/RNA/TNA extraction from cultured cells and tissue using the carboxylated speed beads and guanidine buffers. The only time I see clumping is when there is a lot of DNA/RNA/TNA due to excess input material. What I assume happens is that instead of the nucleic acid just precipitating to the beads in solution, the beads and nucleic acid form one giant complex (almost as if the nucleic acid ‘captures’ the beads instead of the other way around). This bead complex is now effectively saturated, and unable to collect all of the nucleic acid in the solution, hence one reason why you are not getting the yields you expect to see. There are two things I think you could do to improve this: (1) use less starting material (I suspect you will get less clumping as beads won’t saturate) and (2) scale up your extraction if deep well plate will allow – more beads/volume will mean greater capacity to capture nucleic acid.

GEL EXTRACTION

I am not sure if we ever tried doing PEG/NaCl cleanup from a solution that contains agarose, but I imagine it would make for quite a viscous/slimy solution as you describe…between the PEG crowding agent and melted agarose this is perhaps to be expected.

When using the original protocol (#4.3), you mention more clumping. Again, we did not see this in our experiments, and I suspect this is part of the reason why you have a lot of salt contamination. The dense clumps tend to trap GITC/salt, and are not easily washed.

Have you tried adding more (TE-diluted) Speed Beads to the solution? The protocol suggests to add 5-10 uL of STOCK SOLUTION ‘BOMB’ beads (i.e. undiluted), however, if you added 5-10 uL of DILUTED Speed Beads, you may not have enough.

Would be great if you can post back the result so we can help other users trying to use speed beads for plasmid/gel extraction.

Cheers,
Tim

[Author]

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