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?
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.
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.
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.
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.
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.
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.