This case study evaluates the plasmid yield of pcDNA3.1(+) using the AmMag™ 1100M system under different experimental conditions, including variations in culture media (LB, TB, and Plasmid+) and culture volumes. The study aims to identify the optimal conditions for maximizing yield while ensuring consistency and reliability across protocols.
Experimental Conditions:
Plasmid: pcDNA3.1(+) with ampicillin resistance
Host Cells: E. coli DH5α
Culture Media: Lysogeny Broth (LB), Terrific Broth (TB), and Plasmid+
Culture Volume: 1–2.2 mL, based on Optical Density Volume (OD600 × Volume, ODV)
LB: ODV <18
TB and Plasmid+: ODV 14-24
Growth Conditions: 16 hours at 37°C, 800 rpm
Purification Kit: Low-endo (L01037)
Customized Protocols for Different Media Types with AmMag™ Mini 1100
Figure 1. Protocol used for bacterial grown with LB Culture
Media at ODV <18.
Figure 2. Protocol used for bacterial grown with TB or Plasmid+
Culture Media at ODV 14-24.
Plasmid Yield Across Media Types with the AmMag™ 1100M
Figure 3. Plasmid yield data for all plasmids purified with the AmMag™ Mini 1100 across LB, TB, and Plasmid+ media indicates that the optimal bacterial culture volume is approximately 2.0 mL. High-copy plasmid yields can reach up to 18 µg with LB, 38 µg with Plasmid+, and 50 µg with TB media.
Plasmid Purity Across Media Types: A260/280 and A260/230 Ratios
Figure 4. Consistency of Nucleic Acid Purity Across Media Types as Measured by A260/280 Ratios for Plasmids Purified Using AmMag™ Mini 1100.
Figure 5. Evaluation of Protein Contamination Levels Across Media Types as Measured by A260/230 Ratios for Plasmids Purified Using AmMag™ Mini 1100.
Plasmid Purity Across Media Types: Endotoxin Levels
Figure 6. QC data shows that endotoxin levels for all plasmids grown in LB media and purified with the AmMag™ Mini 1100 are below 0.1 EU/µg.
Figure 7. QC data shows that endotoxin levels for all plasmids grown in TB media and purified with the AmMag™ Mini 1100 are below 1 EU/µg.
Conclusion:
The AmMag™ 1100M system demonstrates its capacity for high plasmid yields, particularly when using TB and Plasmid+ media, reaching up to 50 µg and 38 µg, respectively, at optimal culture volumes (~2.0 mL). The results indicate that yield is significantly influenced by media choice and culture volume, emphasizing the need to adjust protocols based on Optical Density Volume (ODV). These findings highlight the AmMag™ 1100M as a reliable, efficient solution for high-copy plasmid purification, supporting its application in research requiring high-quality DNA.
