High-throughput PhyTip® purification of transfection-grade plasmid DNA

Introduction

Therapeutic discovery and screening programmes require diverse plasmid libraries covering antibody candidates, vector designs, and assay components, including antibody-expression and target-expression constructs. Vector libraries may include transfer, packaging, envelope, helper, RepCap, and capsid-variant plasmids for evaluating delivery systems and therapeutic designs.

A robust pDNA purification platform must perform consistently across diverse plasmid sequences used in antibody and vector screening.  High-throughput plasmid purification is therefore essential, enabling tens to hundreds of constructs to be processed in parallel with yield, purity, and consistency suitable for mammalian-cell transfection and therapeutic screening.

In this application  note, we present a high throughput method combining the automation compatible PhyTip® format with Astrea’s DEAE PuraBead® HF resin. This anion exchange resin is well suited for the selective purification of plasmid DNA and removal of RNA, genomic DNA, and endotoxin to provide high-quality plasmid for the transfection of sequences to produce complex biologicals. The resin is a monodisperse chromatography bead capable of running at high flow rates with low back pressure. This highly crosslinked agarose base matrix is made using a sustainable aqueous process and doesn’t contain any persistent plastic components. 

DEAE PhyTip® columns are packed with DEAE PuraBead® HF resin from Astrea Bioseparations. The 1000 µL columns containing 160 µL of resin are available in Tecan, Beckman, Rainin ME, and Hamilton formats. Here we present a manual lysis and clarification method, followed by capture and purification of plasmid DNA using a Biotage ME semi-automated multichannel pipette. In this evaluation, a high copy number plasmid (mCherry) feed was processed using 160 µL DEAE PuraBead® HF packed into 1000 µL PhyTip® columns. A proprietary buffer system was used to support plasmid DNA binding, impurity removal, washing, and elution under automation-compatible conditions. Detailed method parameters are available through Biotage technical support or application consultation.

Workflow

1.    Prepare bacterial cell paste or culture pellet.
2.    Resuspend cells in Tris buffer containing RNase A.
3.    Lyse cells using alkaline lysis buffer.
4.    Neutralise and precipitate debris using precipitation buffer.
5.    Clarify lysate by centrifugation and filtration where required.
6.    Condition the sample with endotoxin removal buffer before PhyTip® column capture.
7.    Run PhyTip® column equilibration, capture, wash, and elution steps.
8.    Analyse plasmid DNA directly after elution or concentrate by alcohol precipitation.

Figure 1. Workflow overview used in method.

 Materials and reagents

• DEAE PhyTip® columns, 1000 µL Rainin ME format, containing 160 µL DEAE PuraBead® HF resin per column; compatible with the Biotage ME system   

• Biotage ME semi-automated purification system including a 12-channel 1 mL pipette, purification software, power supplies, and accessories.

• mCherry plasmid-containing bacterial cell paste, E coli DH5α strain which was transformed with a ~8 kbp mCherry plasmid. 

Process followed inoculation, batch phase till glycerol depletion was indicated; exponential feeding phase then a production feeding phase for 10h prior to harvest. Harvested by centrifugation.

• Resuspension buffer

• Lysis buffer

• Precipitation buffer

• Endotoxin removal buffer

• Elution buffer

• 70% ethanol, neat isopropanol, ultrapure water

• 96 well 20 µm filter plate

• centrifuge, UV-Vis spectrophotometer, and agarose gel electrophoresis equipment

Protocol

The following protocol describes automated parallel purification of plasmid from clarified lysate (Table 1).

1. The PhyTip® columns were prepared by removing the shipping buffer. As 100% glycerol is viscous, this is a critical step for achieving optimal binding. The buffer was removed by aspirating and dispensing 840 µL of water per tip with 3 dual flow cycles and repeating the process using fresh water each time. Using appropriate endotoxin- and RNase-free water for plasmid preparation.

2. Frozen bacterial cell paste/pellet was thawed at 37°C for approximately 20 minutes, then allowed to finish thawing at room temperature.

3. A cell suspension was prepared by bringing 8 g cell paste to 200 mL using equilibration buffer, giving approximately 0.04 g wet cell weight per mL.

4. A 3 mL aliquot of suspension was dispensed into each well of a 24-well plate and centrifuged at 1000 × g for 10 minutes at 4°C to form a pellet.

5. The supernatant was carefully removed. For workflows involving multiple plasmids, pouring was avoided to minimise cross-contamination.

6. Each pellet was resuspended in 200 µL resuspension buffer and mixed until fully dispersed.

7. A 400 µL volume of lysis buffer was added, mixed for 30 seconds, and incubated for 5 minutes.

8. A 400 µL volume of precipitation buffer was added and mixed for 2–3 minutes until viscosity was reduced and a white precipitate formed.

9. The sample was centrifuged at 1000 × g for 5 minutes and clarified supernatant was recovered. Where required, the sample was filtered through a 0.45 µm filter plate.

10. The PhyTip® capture sample was prepared by combining 400 µL clarified lysate with resuspension buffer to 800 µL total volume, then 80 µL endotoxin removal buffer was added immediately before running the method.

11. The PhyTip® method was run using the equilibration, capture, wash, and elution parameters shown below. In this example, 6 tips were used in parallel on the ME automated pipette.

12. Plasmid DNA concentration and purity were measured by UV-Vis immediately after PhyTip® elution.

13. The two elution fractions were pooled where higher total recovery was required.
14. For concentration, an equal volume of neat isopropanol was added, mixed by inversion, and centrifuged at 12,500 × g for 30 minutes at 4°C.
15. The isopropanol was carefully removed and the pellet was washed twice with 500 µL 70% ethanol, with centrifugation at 12,500 × g for 5 minutes at 4°C after each wash.
16. The pellet was air-dried, then resuspended in ultrapure water. Heating at 50°C with rotation improved recovery where plasmid DNA remained associated with the tube wall.
17. Final plasmid DNA was assessed by UV-Vis, endotoxin testing, and agarose gel electrophoresis.

Table 1. Overview of the DEAE PuraBead® HF PhyTip® plasmid purification workflow. Bacterial cell paste is resuspended, lysed, neutralised, clarified, and conditioned before automated PhyTip® capture, wash, and elution on the Biotage ME system. The workflow combines manual upstream sample preparation with automated plasmid DNA purification to support parallel processing of multiple constructs.

Results and performance

The automated DEAE PuraBead® HF PhyTip® workflow delivered consistent plasmid DNA recovery across six replicate tips. Post-concentration plasmid yield averaged approximately 113 µg per sample, with UV purity ratios around 1.92 for A260/A280 and above 2.0 for A260/A230, indicating preparation quality suitable for downstream transfection workflows (Table 2). Endotoxin levels were low after concentration, averaging approximately 1.1 EU/µg.

Table 2. Replicate plasmid DNA yield, UV purity ratios, and endotoxin levels following DEAE PuraBead® HF PhyTip® purification. Six PhyTip® purifications were run in parallel and analysed after concentration to assess sample-to-sample consistency, plasmid recovery, UV purity, and residual endotoxin.

Table 3. Summary performance parameters for DEAE PuraBead® HF PhyTip® plasmid purification and post-concentration recovery. The table summarises key workflow outputs, including yield, final concentration, endotoxin level, UV purity, approximate processing time, and sample input conditions.

Conclusion

Purabead P6HF DEAE resin in the PhyTip® format offers a practical, automation-ready solution for laboratories that need to accelerate plasmid preparation without compromising quality. The method delivered consistent plasmid recovery across replicate tips, with post-concentration yields averaging 113µg of plasmid and UV purity ratios aligned with requirements for downstream transfection workflows.

By combining simple upstream lysis and clarification with automated capture, wash, and elution steps, the workflow helps reduce hands-on time, improve consistency, and support the parallel purification of multiple plasmid constructs. Compatibility with major liquid-handling platforms makes the approach well suited to laboratories scaling antibody expression, vector production, and cell-based screening activities.

Overall, DEAE PhyTip® columns packed with DEAE PuraBead® HF resin provide a scalable and reproducible plasmid purification option for discovery and development teams seeking faster turnaround, reliable sample quality, and an efficient route from construct generation to functional biological testing.

Key findings

• This workflow produced consistent plasmid recovery across six parallel PhyTip® purifications.

• Average post-concentration yield was approximately 113 µg per sample, with final concentrations around 1.2 µg/µL.

• UV purity ratios were consistent with transfection-grade plasmid preparation, with A260/A280 around 1.92 and A260/A230 above 2.0.

• Endotoxin levels after concentration averaged approximately 1.1 EU/µg.

• Our method combines manual lysis with automated PhyTip® capture, wash, and elution steps to support higher-throughput plasmid preparation for antibody expression, vector production, and screening workflows.
  
  

Literature number: AN1040