Kaziya Lee, Applications Specialist, demonstrates the importance of using an appropriate test solution when optimizing a liquid handler to dispense master mix.
The Artel MVS Multichannel Verification System, PCRMix solution, and a Hamilton STARlet automated liquid handler (ALH) were used for this experiment.
Master mix is a critical component in any PCR-based assay such as qPCR and NGS library prep. However, it behaves very differently compared to regular aqueous solutions, as shown in the image below, and different liquid class settings are required for accurate volume delivery.
Aqueous vs. PCRMix Comparison
Just like real PCR master mix, PCRMix QualAssure is more viscous than regular aqueous solutions and clings to the inside of the tip, making it challenging to pipette accurately.
As shown in the graph below, an automated liquid handling method was optimized to transfer 9.9 µL of master mix, then the same method was used to transfer a regular aqueous solution. The resulting volume was approximately 3 µL.
The amount of master mix added to your reaction can directly affect a qPCR assay’s Cq value, as seen in the graph below, which correlates to the number of cycles needed to detect a real signal from a sample. That’s why it is important to ensure that your liquid handler is dispensing the expected volume of master mix.
Comparison to Commercial Master Mixes?
The new Artel PCRMix solution has been formulated to mimic the liquid handling properties of a variety of commercially available master mixes, which can be seen here in this comparison graph. Additionally, using a mimic solution allows you to simulate and optimize your dispensing steps without wasting expensive reagents.
The Artel MVS and the PCRMix solution was used to dial in a 9.9 µL transfer using a Hamilton STARlet ALH.
The optimization process can be broken down into 4 steps: dispense, measure, assess, and adjust if needed. These steps are then repeated until the performance has been dialed in successfully.
First, use the Hamilton STARlet to dispense three replicates of 9.9 µL of test solution into the 96-well plate.
Next, measure the exact volumes that are dispensed into each well using the MVS.
Assess the results by reviewing the MVS output report which shows the accuracy and variability of each dispense, this allows you to evaluate if any changes need to be made. If the %CV is relatively high, as shown here at over 6%, some of the liquid handling parameters may need to be adjusted.
Changes to aspiration speed, dispense speed, and blowout volume can be made to improve repeatability.
In this case, after decreasing the dispense speed and increasing the blowout volume, we were able to improve the %CV from over 6% to less than 1%.
However, the average volume is still low by about 12%. In order to improve the accuracy, we adjusted the liquid handler’s correction curve to compensate for the offset
Because the dispense was low by approximately 1.2 µL, we added 1.2 µL to the correction curve value as seen below for a target volume of 9.9 µL, and then retested.
After adjusting the correction curve, the optimal performance was achieved with high accuracy and low variability as demonstrated in the graph below.
By testing and adjusting your liquid handler with a solution that properly simulates your master mix transfer step, you will be able to ensure accurate pipetting and reliable results.
If you would like more information on how we can help support your automated genomic workflows, don’t hesitate to reach out. We’re here to help.