CLLD not working with ABI PCR Plate

Hello,
I have encountered a strange behavior with ABI PCR plate (cat#N8010560). During aspiration, If the volume in the well is greater than 75uL, the channels have no issue detecting the liquid. However, when the liquid is 50 or below, the channels hit the bottom of the plate and throw a liquid level not detected error, during the aspiration. The issue was really isolated to this PCR plate. The plate is made of polypropylene, so it should be not be any issue with conductance. I am trying to aspirate 25ul out the 50ul.

Anyone has encountered an issue using these plates?
Thanks,

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Hi there,
in the manual it says:
image

So your volume is close to the detection limit. What I’d try to do: Increase the sensitivity or, if this does not help customize your error handling in case the liquid is not detected so that the aspiration is done at the bottom of the tube.

best
Dominik

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we had the similar situation. we just wrote a new defination which can detect till 20ul. if you want i am happy to share that.

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Thanks Kalpesh,
If you dont mind sharing that would be great. I played with the definition and did not manage to get it to work yet.
Cheers,

Hi @Starrif2263,

What kind of carrier is the PCR plate sitting on? If it’s on one of the grey-tabbed carriers, it is likely that the bottom of the wells aren’t making contact with the metal plate. This is a known possibility which varies from labware to labware depending on the height difference between the bottom of the well and the bottom of the plate. This lack of contact decreases the effectiveness of cLLD, requiring increased sensitivity in the pipetting commands. Alternatively, you can move the PCR plate to one of our PCR carrier positions or put a metal plate adapter/chilling plate underneath the PCR plate for improved performance.

Thank you,
Dan

Hi Dan,
Thanks for the suggestion.
I am not using any of the PCR support base. I used a regular 96 corning plate as a plate holder for the half skirted ABI plate. I have also used a 3D printed holder for the same ABI plate. In both cases, I am noticing the same cLLD issue with either holder. But may be a metal holder will be more effective at establishing the conductance. I will give it a trial.
Thank again,

I’d like to see this, too! I’ve encountered the same issue before and couldn’t change it to detect with less volume

Hello All,

It seems i can not attach the defination here. let me know if there is any other way to share the files.

Hi Kalpesh,
Can you attached screenshots? Or you can add the rack an container in the folder that Eric created for users on this forum to share files.

Cheers,

is the error liquid not detected or insufficient volume? I’ve had success with changing well definitions when encountering insufficient volume errors…especially v-bottom and pcr plates changing the lower segment by increasing the lower diameter really helped out with detecting sufficient volume to then do an aspirate.

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All good points:
Make sure wells are touching a metal plate
Adjust the cLLD sensitivity
Tips for 3D printing, use a conductive filament.
https://a.co/d/hvtnSKY

Could be on the hardware side:
cLLD sendor
cLLD bronze cable could be damaged or dirty or old
the washer could be dirty

If all 8 are failing, its not likely the hardware.

when you sort it out, definatly share the solution!

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how much would you increase the inner diameter?

completely depends on the labware, the volume remaining physically in the plate, the dead volume for the aspiration, the aspiration offset if there is one once liquid is detected. I experimented with my case and found that increasing by 4-5mm achieved the results I was looking for, but each case will be different.

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What is important to know is, what the goal of the application is and what volume you would like to aspirate from the available. I have recently gone down the rabbit hole to optimize detection, liquid following, dead volume and the like.
Several things I have learned in the process:
-the volume calculation is completely unreliable. Thus an “insufficient” error may be thrown, that is not in any way justified.
-Liquid following will routinely not be fast enough in conical wells.
-The immersion depth of the tips does reduce the calculated available liquid. You can virtually set the labware lower but avoid crashes with setting the “maximum pipetting height” accordingly.
-the liquid level height is what I am now working with. I will handle the error according to the detected height. As long as I know from measuring, that the volume is sufficient, I will ignore the error and just aspirate from bottom.
-I have set up a test method where small amounts of liquid are removed and the liquid level measured. This enables to measure the true available volume. If anyone would like to have it, just let me know.

Yeah that would be really great to check out and I can see it being really useful. I’m interested :slight_smile:

Sure, I’d rather share via e-mail.

We had a similar problem using 3D-printed adapters. I will check conductive filament as well. But the quick fix was so stupid that it worked: aluminum foil. I’ll make it prettier, as this was a proof of concept, but the effect is remarkable.

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Does the foil extend down to the deck or is just wrapping the nests where the plates sit?

It connects the surface to the aluminum of the support - thus it probably creates a connection between the deck and the outside bottom of the wells. In my test I had 22 detection errors (no liquid detected) without the foil and zero using it.

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This is amazing and so simple! I gave up on low volume LLD long ago but may return. What was the minimum volume you could use LLD. Or did you stick with 50uL?