Using a gripper to lift a plate and put it in a magnet
Using magnetic rods to move beads between sample, like kingfisher does
Could someone please comment on what is the most reproducible way to automate magnetic bead handling in different volumes (I need 20ul - 2ml volumes in different viscosity solvents)? E.g. I know that approach #1 was very irreproducible on opentrons and I hear approach #3 works very well on kingfisher. I have tried approach #2 and it works very well in small volumes.
But has anyone tried approach #2 with large volumes?
Main pros and cons as I see them, related to your needs:
Type 1: Actuated magnet position
Pro: No plate movement necessary, small labware footprint
Con: Typically lower strength magnetic field, optimized for mechanics of actuating the magnetic platform rather than performance
Type 2: Magnet plate + gripper
Pro: Most agnostic approach in terms of technique, lots of variability for plate types/volumes/comfort (e.g. bar vs ring magnets)
Con: Require plate movement, distance between magnet/volume can be variable depending on height of liquid column (e.g. deep-well plates/falcon tubes)
Type 3: Magnetic processor
Pro: Typically walk away and preprogrammed, long magnetic rods mean efficient and consistent magnetic stationary phase, low barrier of entry
Con: Usually expensive single-tasker, Typically have volume ranges* (e.g. cant use 20uL and 20mL on the same unit), less optimization possible
*KF Apex would be a exception to this, but I think its like $150k without the heads
Overall for your volumes, you could make any of these options work. 20uL is difficult on a lot of applications using typical 45mm/2mL 96DW plates as it’s typically a smaller volume than covers the bottom curve of the wells.
There’s a couple tricks you can use:
For magnetic plate:
Split reaction 1–>8 which will not use more reagents, but will allow you to use plates that facilitate mixing and magnetization better. You would need to pool samples together prior to elution (typically a wash is good for this purpose, assuming this isn’t a basic cleanup) such that your eluate vol isn’t <5uL. In general, you can expect ~2-5uL of the volume to be sequestered by beads so account for this in your overages for elution.
For magnetic processor:
program more elution vol than actually in use - this allows more efficient mixing at elution as it increases the amplitude range and might assist in elution efficiency.
Of course, there’s also semi-manual protocols where you manually elute if the volume is just too low.
As for your second question, I’ve personally made methods for cfDNA using like 25mL total volume on magnetic plates - so it’s very possible to use that technique for large volumes. for standard 96DW plates it typically just needs good mixing and long magnetization times - 3-5m for good magnets and longer for lower strength magnets.
I’ve worked with both #2 and #3. For larger volumes I vastly prefer the Kingfisher. It is very good at what it does for volumes ~1mL. IMO, the physics of the Kingfisher is superior to an external magnet and you’ll lose less beads when using the Kingfisher with larger volume.
However, for smaller volumes I like #2. I think you can make a more efficient protocol in terms of both timing and volumes of reagent used. Anything less than 50uL I would usually prefer to go the external magnet route and move a plate on/off. It’s that 50uL - 200uL range where there’s a tipping point depending on a lot of factors.
I’ll also say that I’ve been swayed with some tip-based extractions. We currently use beads on a walk-away workcell and if we have problem it is always the beads. Either they didn’t get mixed properly, we ran out, or it clogged something on the device we use to dispense the beads. We’ve been doing some test with tip-based solutions and although we haven’t fully switched we should be switching away from beads soon.
I think it would be much easier to design a protocol that could handle wide volume ranges with a tip-based extraction rather than with beads and a magnet. I could see using these tips for anything from 20uL to up to 1mL without much trouble.
Yes, uhhh… to be more transparent I was the alpha tester for that product.
DPX approached me trying to sell some other things that I didn’t need. I told them how much I hate mag beads and they said HMB. They came back a few months later with a prototype and testing went really well. I’m excited to put them in our production protocol, they are working on ramping up production. Even today we had a problem with the mag beads on our workcell.
Good point. There’s definitely instruments on the market in the sub $50k or even $30k price range, but lack a lot of the benefits of the KF software. Heck you can even get Maxwells off Ebay for like $1000 but making custom scripts would be much more difficult.
Yes, PCR cleanup. We do a 25uL reaction and elute in 25uL, but both of those could definitely be varied without too much trouble. I think you could pretty easily elute in less than 10uL if you wanted to concentrate your product.
I have done all of these and 1 and 3 are the best. The Opentrons mag module is jenky at best which is likely why your results were poor. The Dynamic Devices iMagZ uses Alpaqua magnets so strength is not an issue. Personally I have moved to a Kingfisher type approach. Its just better.
For PCR clean up we us a C.Wash/G.Pure. It’s WAY better than a KF or liquid handler based system. It uses WAY less plastic and tips. It is a bit $$$ but for us its well worth it. We could not hit our throughput without it.
For extractions we use a KingFisher Flex. Thermo is letting them go for under $35k now and used they can be had for $15k.
Interesting. Do you dispense the beads using the G.Pure? Or just Ethanol and Elution? No matter how the beads are dispense how do you do the initial mix with the PCR reaction? How about the same with the Elution? I assume the plate goes to a liquid handler of some sort to do that step?
We add beads, mix and elute on a Lynx VVP. It’s nice because it only uses 2 boxes of tips. I want to get a DragonFly to add the beads in the future. For elution I do use our D2 because the G.Pure is not very precise but so fast to wash.
I’ve been running the Bluewash and C.Wash for these cleanups as well.
Our team does the elution using them but it can be variable volume wise. Not an issue in our processes but if you’re looking for hyper precise elution. I wouldn’t trust it for beads. This was handled by a Hamilton in a previous workflow and then will be handled by a Mantis (clog-willing) in the future.
Thanks both! We currently use a Mantis to dispense beads and then the plate gets moved to a Bravo for the rest of the cleanup. I have no problems with the Bravo, it’s the Mantis and the beads that give me headaches.
For a completely walk-away solution (as in it’s 1am and the workcell is doing cleanups) we have a lot of problems with dispensing beads.
The Mantis chip can clog if the PEG dries out on the nozzle.
For long runs the tubing that feeds the chip can work it’s way out of the chip.
Same goes for the tubing going into the 50mL conical of beads. It just works it’s way up so that the last plates in the run might not get beads even though there’s plenty in the tube. Our solution for both is lab tape.
We use a V&P Sci Spin Vessel to keep the beads in suspension during long runs. This exacerbates the two former problems. But it also limits us to 50mL conicals, which is about enough beads for 12 96-well plates. We frequently do more plates than that. We also can’t use the continuous flow chips because of this, which would cure those problems.
So yeah, that’s where we are. I think a tip-based cleanup is still a good solution as our main problem is them damned beads.
in most cases, most magbeads are not near saturation. Where a KF type solution shines is not just in the movment with magnets, but the mixing from displacment. This mixing via displacment is better and shaking or asp/disp w tips by quite a bit.
I’ve got a workcell that is coming online where the bead dispenser was intended to be the Mantis. This has been a worry for me as we move towards that portion of the workflow.
Have a few backup plans but the big one is to create the bead plates in advance since we have the storage space for it
Another was to do a quick absorbance measurement on the plate reader to verify beads are being dispensed. Doesn’t help if it’s 1am but it does at least prevent the plate from moving forward.
I might give the CF chip a go - the higher dispense pressure seems like it could help reduce the clogging chance. I’m keeping things resuspended with a vortex genie on an I/O currently so could make this work I think.
