In a separate Knowledge Base article we discuss manual methods for quantitating purified DNA samples. Manual quantitation is perfectly acceptable for small numbers of samples but can become prohibitively burdensome when working with larger sample counts or when conducting frequent, daily, repetitive protocols. In this brief note we discuss how we automate one of our DNA quantitation protocols, used primarily for HLA sequencing and HLA typing.
We use a Qubit dsDNA Broad Range (BR) Assay Kit and protocol in one of the first, and most critical, steps for HLA Next-Generation sequencing. We currently use the Opentrons OT-2 platform for automation of the Qubit protocol. Of course there are many lab automation vendors that can fill this role, but we’ve found Opentrons to be a very cost effective solution for numerous HLA related protocols.
Fig. 1 shows an example of the Opentrons robot as it pertains to the Qubit BR assay. This particular deck layout is designed for up to 22 samples. It could easily be expanded well beyond this but works well for our current HLA protocols. The total runtime for 22 samples is about 15 minutes. The deck layout is optimized to minimize the travel distances of the pipettors, thus shaving off a minute or two of runtime. And the layout is also optimized to minimize the number of pipette tips used per run. Saving plastic reduces costs.
The Qubit BR assay kit includes the following items:
- Qubit dsDNA BR Buffer
- Qubit dsDNA BR Reagent
- Qubit dsDNA BR Standard #1
- Qubit dsDNA BR Standard #2
- Purified sample DNA is added to the assay kit
The Opentrons deck layout includes the following BR assay kit and lab items:
- Slot #1: 10 uL pipette tips
- Slot #2: Qubit assay tubes. 2 tubes for Standards #1 & #2. Remaining tubes for DNA samples.
- Slot #3: 2 mL sample DNA microcentrifuge tubes
- Slot #4: 300 uL pipette tips
- Slot #5: BR Reagent tube, BR Standard #1 tube, BR Standard #2 tube, 2 mL Working Solution microcentrifuge tube
- Slot #6: 5 mL Working Solution microtube
The Qubit BR assay DNA quantitation protocol performs the following steps in order:
- Create Qubit Working Solution
- Distribute 190 uL Working Solution to the Qubit assay Standard tubes.
- Distribute 198 uL Working Solution to the Qubit assay sample DNA tubes.
- Add 10 uL Standard #1 to the Qubit assay Standard #1 tube.
- Add 10 uL Standard #2 to the Qubit assay Standard #2 tube.
- Add 2 uL from each sample DNA tube to its respective Qubit assay sample DNA tube.
When the robot is finished, the Standard #1 & #2 Qubit assay tubes and the sample DNA Qubit assay tubes are manually vortexed, centrifuged and Qubit’ed to determine sample DNA concentration.
This automated Qubit protocol yields accurate and reproducible DNA quantitation results. As part of our QC (quality control) tests, we compared numerous automated Qubit runs side-by-side with manual Qubit methods. The corresponding measured DNA concentrations were within the margin of error for the Qubit fluorometer. Thus, we feel confident that automated DNA quantitation is equivalent to manual quantitation with respect to the accuracy of results.
Furthermore, this automated method significantly reduces the potential for errors that are inherent in manual methods. Well trained, highly experienced lab technicians can certainly perform the Qubit protocols with a high degree of integrity. However, when these very repetitive protocols are applied across hundreds and thousands of samples, mental fatigue can set in, leading to occasional performance errors. These highly repetitive tasks can be performed instead by robots.
Finally, using robots for DNA quantification frees up time for lab techs to perform other, more important, high value added tasks. The accumulated time saved by using this robotic method is significant.