Automation workflow for high-throughput arrayed plasmid DNA preparation and quantification

Area of Science:

• Molecular Biology
• Biotechnology
• Automation Engineering

Background:

• High-throughput plasmid DNA library preparation is crucial for applications like CRISPR screening but is often labor-intensive and costly.
• Variations in plasmid yield and quality can compromise the suitability of entire libraries for downstream experiments.
• Current manual methods are prone to human error and require significant hands-on time.

Purpose of the Study:

• To establish an automated workflow for high-throughput plasmid DNA preparation and quantification.
• To increase productivity and reduce human intervention in generating arrayed plasmid DNA libraries.
• To ensure consistent yield and high quality of plasmid DNA for downstream applications.

Main Methods:

• Utilized the Biomek i7 Hybrid automated workstation for robotic liquid handling and peripheral instrument integration.
• Employed alkaline lysis and magnetic bead purification for plasmid DNA extraction in a 96-well plate format.
• Quantified plasmid DNA using the PicoGreen assay and compared results with NanoDrop UV spectrophotometry.

Main Results:

• The automated workflow achieved median and average yields of approximately 9.5 and 10 µg per sample (7.6-8 µg/mL of culture) via PicoGreen assay.
• NanoDrop measurements indicated higher yields (median ~25 µg/sample, ~20 µg/mL culture) with over 98% of samples exceeding 10 µg/mL.
• Demonstrated a strong positive correlation (R² > 0.8) between PicoGreen and NanoDrop quantification methods.
• DNA gel electrophoresis confirmed high plasmid quality across the generated library.

Conclusions:

• The developed automated workflow provides a robust, scalable, and cost-effective solution for high-throughput arrayed plasmid library generation.
• Automation significantly enhances productivity and consistency in plasmid DNA preparation, mitigating issues associated with manual methods.
• This platform is well-suited for demanding applications requiring large numbers of high-quality plasmid DNA samples.