@tomek
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Dear Sebastian,
Great to hear that you could adopt the protocols. BTW, you don’t need to synthesize the beads in anaerobic conditions. The synthesis protocol works well without argon or N2 environment.
It would be great if we could include midiprep or other protocols.
Cheers,
T.
Dear Guilherme,
Thank you for the question. The protocol 1.1 magnetic core particles are not stable long term. They have to be coated either with silica (protocols 2.1) or carboxyl (protocol 3.1) for full functionality. I guess if you were using COOH beads before, then you could use the #3.1 beads for coupling. Another chemistry would have to be used for silica beads.
Hope this helps,
Best regards,
Tomek
Dear Jonas,
Sorry to hear that it still didn’t work for you. One thing that comes to my mind is that the iron salts could be old. Buying fresh good quality chemicals solved a similar issue for another user. And then the synthesis worked like a charm. Not sure if this is the trouble here, but small amounts of fresh salts one can buy relatively cheaply and if this indeed solves the issue would be great.
Cheers,
T
Dear Jstees,
Thank you for your question. Indeed the concentration of the beads is not stated. We typically check the efficiency of the synthesized magnetic beads (titer the bead amount in the purification procedure and see the outcome), before we apply them for extraction from “real” samples.
After the synthesis, we mix the wet beads with same volume of water (this is our stock stock). From this (or further dilutions in water) we take different amounts for purifications to benchmark the beads. And basically, using this information we take the appropriate concentration of beads for the preps.
We state volume in the protocol, simply because the concentration of the buffers/solutions was optimized (and should not vary too much for preps to work nicely).
Hope this helps,
T.
Dear SilicaFig,
Thank you for your message. Frankly speaking, I did not consider the possibility that TEOS might not be accessible to some, but you are right.
We did not try to use sodium silicates for coating, but it seems it is not too difficult to do, so we might give it a try sometime. If you are planning to try this before we do, it would be great if you could share your experience here for others.
Best regards and thank you,
T.
Hi Joel,
Sorry for the late reply. Thanks, it is in part reverse and forward engineering, but our hope, in the end, is that we made it accessible to everyone who would like to do cheap and efficient nucleic acid extractions using magnetic beads.
Regarding your question, there are quite a few possibilities to conjugate additional molecules to the beads, dependent if you would like to use the carboxyl- or silica-beads. We are in transition now to a new lab, but we will continue developing new protocols for other purposes, like protein purification, or conjugating antibodies to the beads. I will keep you posted once we have something “usable”.
Cheers,
T.
Dear Marco,
Thank you for your question. We did not try other coats than silica and carboxyl (MAA). However, instead of methacrylic acid you should be able to coat the core particles with hydroxyethyl methacrylate (HEMA) in the same kind of polymerisation reaction (simply to replace MAA with HEMA. This should provide a surface with OH groups.
Not sure if this is exactly what you need, but maybe worth a try.
Cheers,
T.
Dear Jonas,
To answer your questions,
1) Yes, from my experience if the ratio is not optimal, one gets precipitate which is also magnetic (not as strong thou) which then in washing gives brownish “color”
2) The solution should be clear and not cloudy. Not sure what exactly the “precipitate” suspension is, but removing them with filtration helps the synthesis
4) We don’t add Tris or any buffer, the only thing what I would do is eventually to add a drop of NaOH to pure water that you use for washing if the pH is too low. Normally, the MNPs should not desolve in H2O during wash and normally we don’t addjust the pH of the water. But, in case your H2O is very acidic, one could consider adjusting it.
Regarding the speed of addition, I don’t think it matters too much here. We add it dropwise, but I also made MNPs by adding the whole iron solution at once. It does change the MNPs size and magnetic properties slightly, but they also work.
I’m glad that you like the protocols, let me know how they worked for you and I’m happy to advise shall you have any more troubles.
Cheers,
T.
Dear Jonas,
Sorry to hear about your troubles with synthesis. We also occasionally had troubles like you are experiencing. There are a few things that are important here.
1) The correct ratio of Fe2+ to Fe3+ (otherwise proper MNPs have no chance to grow properly).
2) It can also be that the chemicals (iron salts) are old. The Fe2/3 solution should be translucent and without precipitates. Either buy fresh salts, or filter the solution. Otherwise, the existing precipitates could influence the MNP formation
3) From our experience, preheating the alkali solution is mostly sufficient to get a good synthesis. Degassing can help a bit too.
4) pH of the washing solution should not be too acidic. Otherwise you simply dissolve the uncoated MNPs.
I hope this helps,
T
Dear Jonas,
Sorry to hear about your troubles with synthesis. We also occasionally had troubles like you are experiencing. There are a few things that are important here.
1) The correct ratio of Fe2+ to Fe3+ (otherwise proper MNPs have no chance to grow properly).
2) It can also be that the chemicals (iron salts) are old. The Fe2/3 solution should be translucent and without precipitates. Either buy fresh salts, or filter the solution. Otherwise, the existing precipitates could influence the MNP formation
3) From our experience, preheating the alkali solution is mostly sufficient to get a good synthesis. Degassing can help a bit too.
4) pH of the washing solution should not be too acidic. Otherwise you simply dissolve the uncoated MNPs.
I hope this helps,
T
Dear William,
There is no need for N2 or Argon! You should degas and heat up the solutions to ~80C and the synthesis should work. Degassing and the heat gets rid of a large part of O2 in the solution and does not disturb the synthesis too much.
Best regards,
Tomek
Dear AKSchiling,
We never tried extracting NA from insects. I suppose this should not be too difficult to optimize. Probably you could follow the “Tissue extraction” protocols, where you would release the NA with ProtK treatment and then GITC buffer for cell lysis and NA capture.
Cheers,
Tomek
Hi,
Not sure if the polarity matters that much. Would need to check. Nevertheless, in our magnetic stands all of them are aligned and have the same pole up.
Tomek
How does the uv/vis spectrum looks like? Do you have shouldering towards 230? We got rid of this problem when we washed once more with EtOH (we did not wash enough and there was residual GuHCl). Potentially it can also be contamination with RNA. Make sure you use fresh (or at least checked) RNase in the buffer. You can check this by running an agarose gel and stain with EtBr, if there is small molecule (~100 bp) band, it would suggest you still have RNA in the sample.
Hope this helps,
Tomek
