I am currently working on a project involving glycerol hit picking using the Hamilton Microlab Prep system and am looking for some insights from anyone who might have experience with this setup. Specifically, I’m interested in utilizing frozen glycerol stocks and am aiming to minimize thaw time during the picking process.
Does anyone have suggestions on effective labware that can be used to keep the plate cold during the picking? Additionally, I am seeking advice on ensuring accurate cell pick-up when the pipette goes into the glycerol plate. If there are particular devices or types of pipette tips that you have found beneficial for this type of application, I would greatly appreciate your recommendations.
Your experiences and any tips you can share would be incredibly helpful as I navigate this part of my project.
While my aspirating/dispensing with cold glycerol stock has been limited to Tecans for the time being, I bet some of my learnings could be beneficial and practical for your setup.
Hey Denise,
The HHC is available for the Prep now! So if you need active cooling, we do have that option for you. If you’d like to explore that, shoot me a DM
A couple of questions to brainstorm ideas to help answer your question:
What is the tube format of your frozen glycerol stocks?
Most people use some form of cryogenic vials (for example), which are a bit wider than standard 1.5mL/2mL “Eppendorf” tubes for which most racks are designed. But cyrogenic vials are often “star-footed”, i.e. they can stand by themselves and can be locked into a holder with a counter-shape to the bottom of the vial. This can make them quite stable, a feature that is very useful for your purpose.
Since you specifically messaged about frozen glycerol stock and “minimize thaw time during the picking process”, I assume you want to wake/thaw a set of specific cell lines (bacterial or eukaryotic) for suspension cell culture from a plate of of many frozen clones/cell llines?
For that purpose, as you already mentioned, you do not want to expose the glycerol stocks to repeated freeze-thaw cycles because it decreases the viability of your stocks.
This means you don’t actually want to aspirate the stocks at all. Ideally you just scrape the frozen stocks with a tip.
This means the problem statements are…
A.) “How do we reliably scrape the frozen glycerol stock to ensure a tiny bit of frozen stock is attached onto the tip that is scraping (while not losing that chunk of glycerol when the tip exits the well/tube, because cross-contamination of stocks)?”
B.) “How do you minimise the thawing of your stocks while the hit/cherry picking of the stocks takes place?”
For B.), consider that any cooling system (I know of) cannot go below 4°C, i.e. is the same as placing your tubes on ice, i.e. they will thaw. Therefore the question is whether any cooler can actually do what you want to do.
My advice is a bit simpler and a bit harder in different ways:
The Prep has a central metal “skirt” on every one of its 8 sites. You can take it out and suddenly have another 50ish mm below of space.
Design your own holder system that neatly snugs into that space, fill it with dry ice, add a holder system on the top that ensures your cryo-vials are always in the exact same position and ask Hamilton to help you create a Prep-compatible definition for it. 3D print or 3rd-party manufacture the little holder.
Pros of this approach: you can have the rack ready tomorrow, it will actually keep your cells frozen for extended periods of time, i.e. allow you to do exactly what want to do.
Cons: dry ice and the prep - you need to be carefull to insulate the holder system to avoid Prep metal deforming in the cold environment that suddenly sits on its deck (not an impossible task but important to consider for the health of your Prep). You can simply print/order a couple of versions of your design with different wall thicknesses and measure the outside temperature of each one.
Depending on your tube dimensions and the possible insulation layer on the walls of the proposed holder the number of samples per Prep site/holder and therefore your throughput might decrease.
From my understanding, this is the only way you will truly minimise freeze-thaw cycles of your stocks. (Little side note: always have 2nd and 3rd copies of your stocks)
Regarding A.), the problem about efficiently scratching your frozen stock with a robot seems a bit harder to me:
If you’d know exactly what the height of the liquid in each stock is, then it’s easy: you just tell the Prep to “pipette” a couple of uL from just below that height.
But if you repeatedly sample from the same stocks, then your scraping will chip away more and more of your stock, moving the known height of the stock further and further down.
I’d recommend testing whether the glycerol stock is identifiable by the Preps conductive liquid level detection (cLLD). I don’t know whether it is but placing a single frozen glycerol stock in a 1.5mL Eppi into the Prep holder for it allows you to very quickly test this out.
If this works then the problem is solved and you just go with Hamilton’s pretty amazing cLLD. If it doesn’t then there are some more complicated ways to think about.
Hope this helps, and please let us know how you are progressing as this is a very useful problem to solve
