Difficulties optimizing aliquoting PCR master mix

Hello all,

I am looking for some inspiration in regards to aliquoting a PCR master mix from a Deep Well plate into PCR plates using the MPH96 on a Hamilton Vantage.

The manufacturer is not allowed to disclose detailed information in regards to the composition of the mix, but it is clear that there is at least surfactant in there which is giving me trouble. Supposedly glycerol content is less than 1%.

I have been working on optimizations but it’s challenging to get the variation within specs. I am trying to get the average volume to 13.5µl, the accepted range is 12-15µl

The set up I have currently is as follows:

Open loop 1 (repeat 12 times)

  • Transport 5 plates to a pipetting position
  • Pick up 50µl tips
  • Aspirate 13.5µl from Source (Prewet)
  • Dispense 13.5µl back into the source

Open Loop 2(perform repeating single dispense 5 times)

  • Aspirate 13.5µl from source
  • Dispense 13.5µl into pipetting position x (1, 2, 3, 4 or 5)

Close loop 2

  • Eject tips
  • Transport plates to rest position

Close loop 1

This is repeated for numerous plates.

The liquid classes that have given me the best performance for the prewet and repeating single dispense are as follows:

I have been testing with a representative liquid, but after using the actual liquid I have noticed performance is worse (5% vs >10% coefficient of variation). I will have to see if I will be able to test with the real liquid, but even if I can I am not sure what my best options are to proceed.

Another problem is that due to the surfactant bubbles will remain in place, which makes a visual check afterwards difficult, which is an important first check to see if the plate can be used later or not. I have been thinking about using a reverse pipetting technique but am wondering about other peoples’ experiences.


Hi Ed,
I am not working with a 96-head, but am also aliquoting PCR Mastermix on a STARlet. I found that a form of reverse-pipetting provided the best results. I settled for aspirating 12µl more than I needed for 18 dispenses of 16µl in a 300 µl tip. The residual volume is discarded.

I always use a set dispensing height without liquid following, that the dispensed liquid will reach during the dispense step to ensure proper transfer. I would thus opt for a surface dispense.

With your settings, the dispense speed slows down to a creep (i.e. the speed is decelerated to meet the stop flow rate). Usually a sudden stop is better to cut off the liquid and overcome adhesion. Air transport volume is quite high. If you don’t need it, which I would estimate you don’t, I would get rid of it. It can be the cause of bubbles, too. If you need it, I would lower the volume - here you are at 10% of your tip volume.

I have no way of measuring the resulting volume besides by eye. Luckily, in a 384 well plate with slightly colored liquid, even minor differences are quite obvious.


Hey Florian,

Thanks for your excellent input. Unfortunately I cannot use 300µl tips as the PCR plates I use are too narrow, which is why I am using 50µl tips with single dispense (and go back to source each time).

I also use a set dispense height without liquid follow, but with jet empty tip. Is there any difference internally/in the way the machine operates if I change to surface dispense?

Your comment about the dispense speed slowing down is also very correct. I have talked to a Hamilton Application Specialist a while ago because I had trouble understanding how the stop flow rate works, he told me not to worry about it, perhaps because I didn’t give enough context. What I do not understand is, how should I set the stop flow rate, is this dependent on my dispense flow rate? Or is there a certain setting which would work with cutting off the dispense fast?

I will also test reducing air transport volume. If I use reverse pipetting, is air transport volume even recommended?

Regarding measuring the volume, I hope the adjustments will reduce bubbles to make it more easy to spot volume differences.
Thanks a lot for your input which already gives me a lot of options to try out.

@Ediagnostics - I would recommend reviewing the following post, which provides an overview of different techniques to use when reusing tips for dispensing tricky liquids:


The second portion of this post goes into further detail regarding how surface dispensing into empty labware, as well as reverse pipetting can be used to reuse tips for tricky dispenses. You will likely want to use a small air transport volume in either case.

I would also recommend leafing through the Hamilton Liquid Handling eBook, which provides a comprehensive overview on liquid handling techniques (surface vs jet dispensing scenarios, starting on page 35) and liquid class parameters (such as stop back flow rate).

Hope this provides additional help.