@emilyjunkins
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Hi all,
We have been able to test this protocol on real samples and see it work, but with less efficiency compared to spin column extraction. We’d like to keep using this protocol for kit-less automation but looking for ways to increase extraction efficiency. Does anyone know if using silica beads vs. carboxylated beads will make a difference, I notice a lot of the mag-kits use silica beads? Would they be interchange able with this protocol?
Any insights would be helpful.
Emily
Hi all,
We are about to try the new BOMB COVID-19 extraction, this is nearly identical to how we prepare for NGS and have good results, so I am very excited to get it working and automated (we’ll share the programming for the opentron system once it gets going). I do want to share though that we added 80mM of DTT to the lysis buffer with GTC before use and saw no more clumping if that is help to anyone on any other protocols.
Thanks for all of the insight and getting this protocol shared!
Hi everyone,
I am working with Bradley Stevenson and wanted to ask a few more questions. We are still using the above procedure with the Sera-Mag prepped beads without PEG. I saw another thread here about bead clumping (specifically carboxylated beads) that suggested it could be both a GTC issue and/or a nucleic acid concentration issue.
1. Your 8.2 protocol uses 40uL beads with 560uL of sample+buffer, when we add 40uL of beads it is to over 1 mL of sample+buffers because of the viral transport medium and lysis buffer (we cant spin down). The thread mentioned above seems to hint at the amount of beads won’t help that issue, but it is something I think is worth a try. Any pointers about bead amounts relative to sample volume?
2. As we worked through out protocol, we had issues towards the end with beads sticking to plastic (low-bind eppendorf) and not interacting with the magnet. We think this could be due to high concentrations of nucleic acids. This is problematic for us since we are trying to set this up for liquid handling systems that won’t be able to scrape beads like we can manually. Have you experienced this?
3. I would like to try your protocol in its entirety but using our prepared carbox beads. Have you encountered any issues adapting this or foresee any when switching to automation?
I realize that we are asking questions about completely different protocols other than the BOMB 8.2. I am hoping you or anyone may have some insight into what might be best for RNA recovery and automation using a magnetic, kitless approach for COVID-19.
Thanks,
Emily
