Automated Low Endotoxin Plasmid Purification Workflow on the Dynamic Devices Lynx

Introduction:

Gram-negative bacteria are common vessels to produce plasmid DNA (pDNA). Endotoxin contamination from the abundant lipopolysaccharides (LPS) in the gram-negative bacterial cell membrane is a major concern when obtaining transfection grade pDNA. LPS often copurify with pDNA, which can reduce transfection efficiencies in mammalian cells. To avoid this, phase-based LPS removal by Triton X-114 combined with anion exchange chromatography is utilized by many commercially available low endotoxin plasmid purification kits. For high throughput laboratories, this process can be time consuming requiring manual interventions using vacuum manifolds. An alternative approach is demonstrated where dispersive solid phase extraction (dSPE) in pipette tips is used to purify pDNA. This concept of dSPE in tips is demonstrated using a Dynamic Devices Lynx automated liquid handler. Using microPure PlasmiTips, 96 low-endotoxin plasmid purifications can be performed simultaneously in 60 minutes.

Methods:

NEB 5a E. coli cells were transformed using a range of plasmid sizes (3.5-8.5 kbp) then cultured overnight in LB Broth (Miller) containing 0.05 mg kanamycin per mL of liquid media. The cells were pelleted and resuspended in 300 µL of 50 mM Tris, 10 mM EDTA, and 100 µg/mL RNAse suspension buffer, pH 8.1. The cells were lysed using 300 µL of 200 mM NaOH, 1% SDS lysis buffer. The mixture was neutralized with 300 µL of 3 M potassium acetate, pH 5.5. After centrifugation, 20% Triton X-114 was added to the supernatant to a final concentration of 2% (v/v). The mixture was transferred to a 96 well plate for processing.

For automated sample processing, Lynx 1200, an automated liquid handling system from Dynamic Devices was used. IMCStips were equilibrated with 50 mM tris, 500 mM NaCl, 15% EtOH buffer at pH 6.5. Following plasmid binding, the IMCStips were washed using the equilibration buffer. The pDNA was eluted in 50 mM tris, 1500 mM NaCl, 15% EtOH buffer, pH 8.5. Purified samples were buffer exchanged using SizeX IMCStips (SEC) for downstream analysis. pDNA recovery was measured using a NanoDrop 2000. A Pierce LAL Chromogenic Endotoxin Quantitation Kit from Thermo Scientific was used following the vendor’s recommended protocol to quantify endotoxin content.

Preliminary Data:

We established an automated low-endotoxin plasmid purification method using a strong anion exchange resin in the IMCStip that enables reproducible pDNA sample preparation from cell lysates. This method is easily integrated onto the Dynamic Devices Lynx platform without any need of additional hardware.

• Average recovery of 4.5 µg of pDNA from 2 mL overnight LB culture (n = 8) and over 10 µg from 2 mL overnight Plasmid+ media (n = 10)

• Average concentration of 100 ng/µL with endotoxin levels < 1 EU/µg plasmid.

• Performed 96 low endotoxin plasmid purifications simultaneously with a processing time of 1 hour.

Novel Aspect:

This automated workflow method can be used to prepare 96 low-endotoxin plasmid preparations using tip-based dSPE on the Dynamic Devices Lynx liquid handling system.