I would like to use the LiHa arm for multipipetting small volumes (1-15uL). However, I am having issues with multi-dispensing. Namely, the first 1 or 2 wells don’t get any volume of liquid dispensed in them.
For example, when I try to aspirate up 143.75uL water from a glass vial and then multi-dispense 28.75uL (i.e., 143.75/5) into 5 wells, the 1st and 2nd wells receive little-to-no volume, suggesting some issue with liquid volume and/or air gaps; the 3rd, 4th, and 5th wells receive the correct volume.
Is this likely an issue with the liquid class? I am currently just using the default “Water free dispense” (see two images below) (I did make one slight change, i.e., slightly decreasing dispensing speed to conform to our LiHa tips bounds; but this class is effectively the same as this default one). Do you have any recommendations for how to make a liquid class work better for multi-dispensing small volumes of water solvent materials? I also tried with ethanol solvent materials, and the same issue persists – any recommendations for multi-dispensing small volumes of ethanol solvent materials?
This is likely just liquid class optimization for specific lab/liquid - but multidispensing is a difficult technique to optimize. The simplest solution here would be to aspirate ~100µL more than require and dispense the first 2-3 aliquots back into the reservoir if your observations are that only the first 2 alqs are affected.
Beyond that, it’s troubleshooting and optimization. Skim over literature for suggestions, such as Tecan’s LC guide, here’s the link from Tecan with multi pipetting in/around section 4-5 and the Hamilton equivalent (still amazing guidance despite different instrumentation) is here - Library Downloads (Links)
Hi @evanc ,
Is this a liquid LiHa with fixed tips and system liquid?
(I assume it since there are no Get/Drop Tips commands in the script)
Then you must add a Wash command (ca. 4ml / use default settings) before and after each pipetting sequence (here: before the Aspirate command and after the last of the Dispense command). With a liquid filled system and fixed tips, if you perform multiple pipetting sequences without a Wash between, the system trailing airgap (STAG) can start to disrupt and negatively impact the pipetting results.
Also perform a Flush/Wash (ca. 40ml) at the start of the day after the instrument has been idle overnight to refresh all the system liquid within the tubings of the system.
We had the issue with empty first column as well, but found the aspiration step was too fast (and you can’t see this because of the black carbon tips). Therefore the last bit in the tip was empty and when pipetting out the first 1 or 2 columns received air or a little bit of volume.
Maybe reduce aspiration speed and see what happens. And maybe you can aspirate from lower point in the reagent
We used AirFCA with Ampure XP Beads when we had the issue.
@DanielYip Thank you all for your tips! Indeed - this is a liquid LiHa with fixed tips.
Adding an initial wash step and decreasing aspiration/dispense speeds as shown in the below image [provided as Google Drive link because I am limited to one media item per post] fixed the pipetting issue as described in my first post. Thank you.
However, for my new script I want to run, it involves even smaller volumes, and I have been having issues. Shown below are my current script and 0.1-15.01 uL liquid class settings (note that the 15.01-200.01 uL settings for Ethanol Free Test v2 are the same as Water Free Test v2 shown above).
When I run this script, wells 2, 3, 4, 5 have approximately half of the desired volume, and well 6 has approximately 1.5x the desired volume, so effectively all wells are problematic. As you can tell in the script, I try pipetting up 50 uL at the beginning and end to flank the actual steps, but this is to no avail. First off, from your experience, can the Tecan LiHa tips (ours go up to 500 uL) even reliably pipette volumes < 10 uL? And more specifically, any tips on how to troubleshoot?
It highly depends on how stringent your liquid classes but we have been able to accurately pipette 1uL with our Tecan EVO and it is on 500uL syringe volume. I don’t think it can do lower, unless you go to 250uL syringe volume.
Have you ensured your plate definitions are correct? Like the bottom of the plate - the position of a z-dispense etc.
You could also try re-tightening the channel tip (the gold part). You should have this small white holder that can lock into the channel tip and you can slowly and carefully tighten it - be careful not to over tighten as that can damage it.
@DanielYip I changed the z-dispense (1580) to be quite close to z-max (1585) to prevent the droplet from not being released into the 96-well plate well. I also tightened the 8 LiHa tips.
The issue still arises; this time - the first well gets the correct volume (i.e., 5.2uL) but the remaining wells get about 4uL.
I tried different aspirating/dispensing with tip #2 or #3, etc., but the same incorrect volume pattern emerged.
I also tried lowering the aspiration and dispense speeds and added a trailing air gap after each dispense (as shown below), but the same problem emerged.
I understand this may be a case of machine-to-machine variability, but for anybody who does have a working small volume multipipetting class, do you mind sharing with me images of the liquid class settings? This would be tremendously helpful as I try to isolate this problem! Thanks!
Following up on this, as @evwolfson suggested, the problem is likely liquid specific. It’s actually not so much a small volume problem, but rather a problem with ethanol liquids.
I am able to accurately dispense 5uL and 20uL of water into each well, but I cannot accurately dispense ethanol. When I try dispensing 20uL of ethanol into a well, it dispenses about 18uL. When I try dispensing 5uL of ethanol, it dispenses about 3 uL; thus, suggesting an error of about 2uL.
Thus, does anyone have advice for working with organic solvents like ethanol? Liquid class suggestions would be appreciated.
@luisvillaautomata Thank you for recommending adjusting the settings under “Calibration”. I am playing around with the “Offset” and “Factor” values now and believe this will get me to a solution (so thank you in advance!).
As a clarifying question, I noticed that when I modify the offset value from 0 to 3.4, my script now produces the error shown in the photo below, i.e., there is insufficient volume by 12.1uL. How does this missing 12.1uL volume come about due to the new offset of 3.4? 12.1 is not divisible by 3.4 so I was not sure.
Here I believe the reason you are seeing this dispense volume issue is because you have a single aspirate command followed by multiple dispense commands back-to-back. The system looks at both the liquid volumes and the air gaps and likely some of the air gaps change after the dispense and by the time you get to the last dispense, it doesn’t have the numbers available it is looking for. So likely you need to bump up the volume on your aspirate command in order to make this error go away on the last dispense. It’s not necessarily just a simple division of aspirate volume / # of dispenses for determining the dispense volume. Ultimately you can use the numbers in the error to help guide how much you adjust the volume on the aspirate side. Mike Mueller Nucleus Automation Partners
Are these fixed tips? I’m going to have to defer to someone with that experience.
With that said, multi-pipetting is always such a hit or miss. it’s possible that you have an airgap somewhere that’s causing you problems.
Hypothetical here but if I am aspirating 1uL and dispensing 1uL using a 1mL syringe, it should be able to multi-pipette 1000 times right? Well that depends. If your air gaps are 20uL each and say you have 2 airgaps, each multi aliquot actually requires 40 uL’s of reserved space in your syringe for the 1uL dispense because of the additional 2 20uL airgaps. And since we’re talking about dilutors and machinery, that translates into (potentially) 1-2 physical turns of the dilutor syringe. So your new total is the overall syringe size minus the reserved volume for the airgaps. Multi dispensing compounds this math with additional variables like Excess Volume and Conditioning Volume which you have to factor into your aspiration/dispense volume math.
I would just aspirate 120µL before the 5x dispense and return the residual to source or waste afterwards. EtOH is probably not a precious reagent so why worry about µL of loss. Not as fun to be actionable rather than know why the problem happens, but it’s better to revisit these sort of issues as optimization rather than 1st version in most cases.
Other than this, consider that your offset is a flat volume and each of these single aspirate/dispense commands add this flat volume as an adjustment individually. Therefore, your aspirate command adds +[Offset] µL volume while your dispenses EACH also add +[Offset] µL volume. Summarized, you are aspirating 1x[Offset] and dispensing 5x[Offset] and have a discrepancy in available vs needed volume. Work backwards instead and change the aspirate command to 100+5x[Offset] for minimum needed volume, or like mentioned in the first part, just add a flat volume sufficient enough to cover the difference.
