Small volume (2uL) pipetting with 50uL tips

Hi everyone,

I’m trying to set up our MLP for reverse transcription and PCR setup in a library prep protocol. I need to aliquot 2uL reverse transcriptase to half a plate and I’m having a lot of trouble tuning the instrument to perform well. I’d love to be able to multi-dispense, but I need a huge pre-aliquot to avoid empty wells at the start, and even then the volumes vary drastically.

I’ve removed my air transfer volume and now I’m considering tweaking dispense velocity, etc. with which I’m unfamiliar. Are low volume dispenses like this feasible on this platform? I’d love to hear some tips if you have any.

I haven’t played with our prep too much so won’t comment on specific changes. Generically speaking, slowing it down significantly is a good first step compared to an aqueous liquid class.

Alternatively, can you make a master mix and dispense that instead? Can dilute with water as needed to hit your final reaction volume and then do x-uL of your aqueous sample/primers.

This would reduce the viscosity of the transcriptase and make it easier to pipette. Dry dispensing 2uL can be tricky to set up.

Thanks for the response! I’ll continue to tweak dispense volumes, but sadly the reaction is just 2uL transcriptase and 8uL sample. No dilutions possible here!

A few quick thoughts:
–We do not recommend multidispensing volumes <15uL. For single dispenses, we do have labs using the Prep for 1uL at a time. (You definitely want to make sure you’re dialing in those liquid classes though, like you are)

–Slowing down the speed is a good place to start, like MNewsom said.

–Would you be able to pipette the 8uL of sample into the well first and then add the 2uL of transcriptase? Adding low volumes into liquid tends to help get all of the liquid out of the tip better than adding to air.

–If you can’t spike it into a liquid, try editing the height of the dispense. Set it to dispense above container bottom and set the height to 0 or 1mm above container bottom. Use the surface tension of the plate to help pull the liquid out.

–If you get it to consistently dispense the same volume, but it isn’t the right volume, then move away from the liquid class settings and tweak the correction curve. That will keep the precision the same (liquid class settings) and dial in the accuracy.

2 Likes

Reverse pipetting can sometimes work magic in these dry-contact dispense scenarios. You might try dispensing (made up volumes) 10uL then aspirating 8uL and dispensing excess to the source reservoir.

Otherwise, like has been mentioned before wet-dispense is always preferential, long delays (>=1000ms) after plunger movement, and having sacrificial conditioning/excess volumes will all improve precision.