High-Quality Plasmid DNA Purification in 9 Minutes Following Innovative FastFilter Technology

Introduction

Traditional methods for isolating plasmid DNA often involve several centrifugation steps, making the process cumbersome and time consuming. Omega Bio-tek’s E.Z.N.A.® FastFilter Plasmid DNA Mini Kit is designed to purify high-quality plasmid DNA from up to 5 mL of culture in just 9 minutes. This Kit utilizes the first-of-its-kind FastFilter mini column nested within a HiBind® silica spin column to combine lysate clearance and DNA binding into one simple centrifugation step, skipping the inconvenient cellular debris pelleting and transfer step. The protocol processing time is further reduced by eliminating multiple centrifugation steps without compromising the quality of the purified plasmid. Here, we compare the performance of the Omega Bio-tek’s Kit to comparable kits from other manufacturer’s and show sequence-quality plasmid purification with similar or better processing times based on number of preparations following Omega Bio-tek’s Kit. We elucidate that our kit is capable of handling diverse samples inputs across plasmid types, culture medium, and input culture volume. We also illustrate the downstream suitability of the purified plasmid through sequencing data and restriction digestion analysis. Omega Bio-tek’s E.Z.N.A.® FastFilter Plasmid DNA Mini Kit provides rapid and straightforward solution for plasmid purification that is suitable for a wide variety of downstream applications such as sequencing, restriction enzyme digestion, transformation, PCR, and routine screening.

Methods

pGEM plasmid DNA was isolated from DH5α cultures using the following centrifugation protocol from Omega Bio-tek:

Isolation Protocol using Omega Bio-tek’s E.Z.N.A.® FastFilter Mini Kit

Figure 1. Centrifugation protocol for the isolation of plasmid DNA using Omega Bio-tek’s E.Z.N.A.® FastFilter Mini Kit.

Other competing kits were used to isolate pGEM plasmid DNA according to the manufacturers’ instructions. The processing time for each protocol was measured during the purification process. After isolation, plasmid DNA was eluted at 100 µL and quantified using Thermo Scientific’s NanoDrop™ 2000c system. Plasmid DNA isolated using Omega Bio-tek’s E.Z.N.A.® FastFilter Plasmid DNA Mini Kit was then used in a 5 µL Sanger sequencing reaction which was analyzed using an Applied Biosystems 3730X. Large-sized pVITRO and low copy number pACYC plasmids were also purified using Omega Bio-tek’s Kit and centrifugation protocol to assess the ability to produce high yields across a range of diverse sample input conditions. Finally, pGEM plasmid DNA from overnight bacterial cultures was purified using the E.Z.N.A.® FastFilter Mini Kit and analyzed by restriction digestion with various enzymes.

Results

Comparison of Processing Time from Bacterial Culture

Figure 2. Comparison of Plasmid Purification times (n=4) using kits from Omega Bio-tek, Company Z, or Company Q. The processing time was comparable using kits from Omega Bio-tek and Company Z at minimum culture volume and was lower by 2 min for Omega Bio-tek at maximum volume. The processing times shown are actual timed extractions including all pipetting, centrifugation, and bacterial culture pelleting steps. 

Competitor Analysis of Plasmid DNA Purification

Table 1.  pGEM plsmid was purified DH5α cultures (n=4) using kits from Omega Bio-tek, Company Z, or Company Q following manufacturer’s recommended centrifugation protocol. Purified Plasmid DNA was eluted at 100 µL irrespective of the manufacturing kit used and quantified using Thermo Scientific’s NanoDrop™ 200c system. High Yield and Quality are not sacrificed for faster processing times.

High-Quality Plasmid DNA Suitable for Sequencing Reactions

Table 2. Plasmid DNA purified using the E.Z.N.A.® FastFilter Plasmid DNA Mini Kit was used in a 5 µL Sanger sequencing reaction. The sequencing reaction was analyzed on an Applied Biosystems 3730XL. The samples had an average CRL of 994 bp and an average QV20+ of 985 with a quality score greater than 20 (≤ 1% probability of error in base calling).

Complete Digestion with Various Restriction Enzymes

Figure 3. pGEM Plasmid DNA from overnight bacterial cultures was purified using the E.Z.N.A.® FastFilter Plasmid DNA Mini Kit and analyzed by restriction digestion. Complete digestion was observed for the various restriction enzymes tested.

High Yield Across Various Plasmid Types and Culture Media

Figure 4. The overlay showed that the purified DNA contained more cfDNA and minimal genomic DNA contamination. Again, the average size of the cfDNA peaks was similar between the manual and automated protocols, with the automated protocol showing a higher intensity around the average peak of 194 bp.

Conclusions

  • Processing time is greatly reduced when compared to competing kits.
  • High yield and quality are not sacrificed for reduced processing time.
  • The purified plasmid DNA is of suitable quality for sequencing reactions.
  • The kit can isolate high yield DNA across diverse plasmid types, culture medium, and input culture volume.

WP-0050

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