I inherited the responsibility of our Tecan Fluent. I am not an expert on automation but I’m having a good time giving it a go. I have lots of questions/troubleshooting needs but currently I am struggling with an FCA Reagent Distribution issue.
I am trying to have the FCA distribute 23ul of liquid in all wells of a 96 well plate. The FCA is pulling the distribution liquid from a single column. I am using all 8 FCA tips. Each well in the source plate has 300ul of liquid. It fills the first 6 columns just fine. It then has to return to the source wells to get more liquid for the remaining 6 columns. Each time, columns 7 and 8 have no liquid in them once it’s done but the source plate is empty. There should be plenty of liquid for this distribution (I technically only need 276ul and it has 300ul).
Unfortunately, we are using black tips, so I cannot see if there is an air bubble. It is not throwing a liquid detection error. There should be more than enough liquid for the distribution.
I think I need to alter something within the liquid class but I am not sure where to start. I would love any advice on where to look for the solution. Sadly, the Tecan manual is not very helpful for this issue.
Multi-dispenses are notorious for using a lot of liquid, and thus your % extra aspiration should be adjusted accordingly. Are you using 350uL (orange) tips? FYI, there are manufacturers out there that sell clear tips, although cLLD would not be functional. Depending on your assay, if you always know you’ll have 300uL present, and the liquid viscosity is low (and won’t cling to the exterior of the tip) within the liquid class I would shut off cLLD and just have it aspirate at zMAX. This will speed up the process. You may also want to check your actual dispense volume to verify you are getting 23uL. If you are using a default liquid class provided by TECAN, whereby it is “Read only”, try duplicating that LC and make sure that both the aspiration and dispense LC formulas for “Accuracy adjustment” are set at zero, then adjust accordingly.
I made these changes but am still having an issue. I also adjusted the amount it was distributing and that helped. The FCA, using the 200ul tips, is now able to fill columns 1-7 on the first source pull. On the second source pull, it is not filling the first column (column 8). I vaguely remember there being a setting you could adjust to make sure it filled the first wells/column on a multidispense. Does that sound familiar at all to you? I’ve also attached photos of two places where that setting might be.
I agree with @Liam7b, the complex liquid handling steps designed to make programming simpler often make your life more difficult in the long run! You have more control of you build it up from basic commands. However, I don’t think that is the case here.
From initial impressions of details you have provided it sounds like there isn’t enough volume left if the source plate for the 2nd lot of dispensing. I’m not sure what plate type you are loading from (pcr or flat bottomed) but 28uL isn’t a lot of dead volume. I belive that the multi dispenser liquid class you use aspirates more than required leaving some residual volume in the tip (e.g. 15uL). Its been a while but i think that this volume might be dispensed to waste when resetting the tips for the 2nd dispense. You can change where it dispenses this to in the settings on the 2nd picture on the right.
This might help sort some of the missing volume problem.
Hi, can you clarify a few details and I should be able to help you out with this. You mention columns 7 and 8 not getting liquid but that you are trying to fill the entire 96-well plate. I would assume you are also not getting liquid in columns 9, 10, 11, and 12, is that correct? Are you by chance getting it to fill columns 9, 10, 11 and 12 but just not 7 and 8? If so, that would suggest something else related to the air gaps so would be good to clarify that.
So you have a 200 uL filtered tip and it’s setup to do multi-dispense, so you have 23*6 = 138 uL dispensed per tip in the first pass - and it picks up extra liquid related to the multi-dispense, and your tip capacity is less than 200 uL because of the filter, maybe closer to 180 uL or so.
If you pause it after it does the second aspirate (for columns 6-12), is there any liquid remaining in the source plate?
Can you post a screenshot of your liquid class details? Not just the microscript but the Aspriate / Dispense Easy Control settings.
One other thing you might consider trying (for testing purposes), it is break this up into two commands. Do one reagent distribution for columns 1-6, then the 2nd for columns 7-12. Put an Empty Tip Contents between the two. See if it behaves any differently. It will use 2 sets of tips instead of 1 but might provide some insight - the empty tip contents will reset the tip between the two transfers potentially in a somewhat different way than when it does it all in one command (would have to dig deeper into the microscript to know for sure).
I think you may want the field for # of samples to be 96 rather than 8.
I’ve attached the easy control pictures for aspirate and dispense below.
It IS filling 9,10,11,12 but not 7 and 8. You mentioning air gaps jogs my memory and I think this is a setting I need to change to fix this. I’ve messed with the trailing air gap setting but that hasn’t improved the issue.
There is liquid remaining in the source plate and the tips are also depositing a fair amount of liquid in the waste plate after dispensing.
Hi @Snobles, when you open your liquid class for viewing, you want to make sure the correct arm type and tip type are selected (top left corner) when making modifications. If you are making modifications to the wrong arm or tip type, your changes will have no effect at runtime.
You’ll notice in some of your screenshots of your liquid class that you have the liquid FCA selected, though your instrument is equipped with an AirFCA. This is a common issue, and newer versions of FluentControl will automatically select one of the arms that is equipped on the system instead of defaulting to the liquid FCA.
Since you have just recently become responsible for the Fluent, have you had an opportunity to attend Tecan’s FluentControl training course? We offer a weeklong training, either remote or in person at our Tecan US headquarters in Raleigh, NC.
Do you see the same behavior when using the default “Water Free Multi” liquid class? Are you able to share the reagent you are distributing to the plate? Tecan may have already developed liquid classes for the reagent you are pipetting, which could give you a head start on your own development and optimizations.
Also, I would be happy to connect you with your local service and applications team.
Thanks for this information. Since it is filling columns 9, 10, 11, and 12, that suggests the following to me…the way you have it setup, it grabs liquid, enough to fill 6 columns, and does the multi-dispense across the 6 columns. Since it isn’t dispensing liquid to columns 7 and 8, but is for columns 9, 10, 11 and 12, that suggests it is dispensing air to columns 7 and 8 - when the air runs out then the liquid starts dispensing for columns 9, 10, 11 and 12. This suggests the air is accumulating in the tip after the first transfer and the tip isn’t being “reset” between the two aspirates. You can reset the tip with the “Empty Tip” command between the two sets, which is the suggestion I made about breaking it up into two steps.
For the reagent distribution command, you don’t have independent control of the tip pickup - it picks up tips at the beginning of the command and drops tips at the end of the command. So if you want it to do this with one set of tips using this command, you have to manipulate the air gaps in the liquid class settings (play around with reducing them to eliminate the air in the tip).
Another approach is to script it without using the reagent distribution command. This allows you to have total control of the tip handling so you can do this with a single set of tips, you can reset the air gap between the two sets of transfers, and should provide the result you are looking for.
