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An update – I have tested the silica-coated NPs in DNA isolation and they worked very well. So to summarize my experiment: to avoid the desintegration of MNPs during washing with water after the synthesis, I’ve pelleted them with magnet, washed three times in small volume of ethanol (instead of water) and then immediately proceeded to coating with TEOS.
Hope that helps, it would be great if someone could confirm that this is repeatable 😉
Gosia
Hi,
I have successfully synthesized the NPs once but I was never lucky enough to repeat the procedure. I have the same problem as reported in this thread – the NPs during washing with water start to desintegrate, the supernatant is getting brown (or even reddish) and the leftover NPs are becoming less paramagnetic. It seems that, at least in my hands, the whole problem is the washing step so I thought that maybe it can be ommited? Because I coat the beeds soon after the synthesis with TEOS and this procedure uses NH4OH, I thought that maybe extensive washing of the beeds and bringing them to pH7.0 is not so important.
So what I did: I’ve settled the NPs after the synthesis with the magnet and washed few times in ethanol (not large volumes – ~10 ml per wash, but please note that my synthesis reaction is 20 times smaller than the original procedure) and added the beads immediately after that to the warm ethanol/NH4OH solution (according to the procedure of TEOS coating). The NPs are still black and the solution did not change color. The NPs are still paramagnetic. I’ll let you know about the results of the coating and DNA binding but so far it looks promising 🙂
Cheers – Gosia
Hi,
have you ever tried to use the nucleotide removal protocol to cleanup sequencing reactions? There some commercial kits utilizing magnetic beads for this purpose and I was wondering if we could be able to deal with as well in the BOMB system
Best – Gosia
Hi everyone,
I’ve just produced silica-coated MNPs. In my first attempt I’ve tried to do it in the same scale as in the protocol but got poor results – the MNPs lost their magnetic properties during washes. I’ve scaled down the procedure 4x and it worked very good. I’ve coated approx. 50% of the beads with TEOS with overnight incubation (the procedure was scaled down by 10x as compared to the original one). I’ve tested the beads in plasmid isolation and genomic DNA isolation from tissues – in both cases the MNPs worked very well (I’ve checked the DNA in electrophoresis and PCR, respectively). Here are some of my thoughts and tips that you may find useful:
– I think that the first attempt of the synthesis failed because I was not able to control the temperature of the NaOH solution. The heating plate of my magnetic stirrer was not efficient enough.
– I’ve filtered the FeCl2/FeCl3 solution, but it seems that the salts themselves do not have to be the highest quality – I’ve decided to use cheap chemicals from a local company and they worked very well
-I’ve degassed the NaOH solution using ultrasound bath instead of vacuum pump.
– The ethanol used in TEOS-coating indeed does not have to be 99%. We have used 96% technical grade ethanol. Tomek, thanks for the tip.
The BOMB project is an awesome idea and I’m very grateful that you decided to share your protocols with the rest of the world. Great work!
Cheers – Gosia