We had our Flex installed in February, but as yet I don’t have any protocols running reliably.
I’m interested in hearing if anyone has run the following validated protocols with success “out of the box”.
In order of expected difficulty, these are the ones I’d like to run.
- DNA Normalization with Flex 1-channel or 8-channel pipette (https://library.opentrons.com/p/dna-normalization-flex-pipettes)
- AMPure XP 48x (https://library.opentrons.com/p/AMPure-XP-48x)
- Illumina DNA PCR-Free Prep (https://library.opentrons.com/p/Illumina-DNA-Prep-PCR-Free)
- Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (https://library.opentrons.com/p/Illumina-Stranded_total_RNA_Ribo-Zero)
The Normalization protocol fails to send to the Flex without errors since I updated to 8.4.0, prior to that it would send but I never had a chance to run it.
The AMPure protocol I thought would be such as easy one to get right but that also fails. Sometimes the wells of clean DNA are empty, other times they still appear to have beads still in there. It’s also incredibly slow at near 2 hours for 8 samples. The default 30 minute shaking twice is excessive, and i’m not sure why it’s adding beads twice.
In fairness to Opentrons they have been making modifications to the DNA PCR-Free protocol after I had failures, but it does have a blue check mark so I’d assumed it would just work. The most recent failures are where in a strip of 8 reactions, all wells of final library were empty except one in the middle of the strip, and that had the wrong volume in it. To get it to work i’m told to sit and watch it to see where it loses the sample. This is a relatively short protocol so that’s easily done, but is it truly validated if a user has to do this?
The Stranded Total RNA Prep is one protocol that i’m trying to build up confidence to automate, but it just feels like i’m a million miles away from being able to start a run and walk away from it. If I have to sit and watch this like a hawk for 2 days to check it completes i’m going to lose my mind.
I still have my protocol development credit to use, but I don’t really think I want to use it up to get a “validated” protocol to work reliably.
Please let me know if you’ve used any of these protocols out of the box and had good results.
Hey Mat,
Happy to have you save that protocol development and help walk you through these at no cost. Please reach out via email to get this started: Meghan.ferzoco@opentrons.com
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Hi Meghan,
Thanks for the reply, and the emails we’ve had back and forth since.
I appreciate the efforts you’re putting in to help us utilize the Flex in our lab.
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Hey there @kellogg76
I find opentrons truly shines when you take the time to customize the protocols for your specific lab. A good rule of thumb is that the protocols they have on the website should be viewed more as templates than as “this will work out of the box 100%”. Opentrons is fantastic because of the open-source python compatible interface, but if you’re not comfortable with that I can see it being a slog
If you’re having issues I’d be happy to take a look with you or discuss if you send me a DM. I personally love my flex
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Hi, apologies for the late reply, I was giving Opentrons time to try to get to the bottom of my issues.
Since I posted in June 2025, Opentrons offered to perform an onsite visit which helped a great deal.
Since that visit we now have the protocol for the Illumina DNA PCR Free kit working.
The Illumina Stranded Total RNA protocol is close to working, with just some small issues to work out.
The yields in both are not quite as good as manually performing them but it’s good enough.
The “DNA Normalization with Flex 1-channel or 8-channel pipette” protocol still gives errors when I try to run it.
These three protocols cover most of what I’d like the Flex to do routinely, and using them successfully will eventually help grow confidence in the platform and allow me to try other protocols.
I think the Flex is capable, but I do take issue with many of the protocols having a verified symbol if they don’t work out of the box even when you use Opentrons consumables.
The marketing very much implies that this is a plug-and-play instrument but I’m not seeing that to be true. The DNA normalization protocol for example is about as vanilla as you can get, as it only uses the pipettes but it fails with an API error every single time and no solution as yet from Opentrons.
We’re now close to one year of ownership and it’s been a bumpy road to say the least.
Just out of interest, what protocols do you use on your Flex, and did you have to do much tweaking to get them running?
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