The EXTRACTME PLASMID MIDI kit (EM16) and EXTRACTME PLASMID MIDI ENDOTOXIN-FREE kit (EM17) with Plasmid Filtration Columns (EM20) are designed for the efficient purification of high quality plasmid DNA from 50-150 ml of cultured bacterial cells. The product is intended for research use only.
The EXTRACTME PLASMID MIDI kit (EM16) and EXTRACTME PLASMID MIDI ENDOTOXIN-FREE kit (EM17) with Plasmid Filtration Columns (EM20) are designed for the efficient purification of high quality plasmid DNA from 50-150 ml of cultured bacterial cells. The kits are based on anion-exchange resins, allowing extraction of ultrapure, transfection-grade pDNA, which is highly suited for use in demanding applications. The isolation protocol and buffer formulations were optimized for high isolation efficiency and pDNA purity. The product is intended for research use only.
The plasmid DNA purification procedure utilizes pre-packed anion-exchange resin columns which efficiently and selectively bind nucleic acids. In the first isolation step, the pDNA is released from bacterial cells by alkaline lysis. Then the lysate is neutralized and all the cell residues along with the proteins and genomic DNA are separated in the centrifugation step. This alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. In the next step the lysate is applied to the purification column with the equilibrated resin and the DNA is bound. The washing stage effectively removes impurities and enzyme inhibitors. A suitable buffer with a high ionic strength allows the elution of the plasmid DNA, which is then concentrated and desalted by precipitation. The purified plasmid DNA may be used directly in all downstream applications such as PCR, qPCR, transfection, microinjection, Southern blotting, DNA sequencing, enzymatic restriction and so forth, or stored until ready to use.
Bacterial broth culture:
50-100 ml (high-copy number plasmids)
100-150 ml (medium- and low-copy number plasmids)
100-500 μg of transfection-grade pDNA from 100 ml of cultured bacterial cells
- 50 min for EM16 and 80 min for EM17
- additional ~60 min for DNA precipitation
A260/A280 ratio = 1.7 – 1.9
ENDOTOXIN REMOVAL (EM17)
<0.1 EU/μg verified by LAL
RECOMMENDATIONS AND IMPORTANT NOTES
Plasmid Filtration Columns (EM20) – Discontinued - Manual
Plasmid Filtration Columns – TROUBLESHOOTING
The quality of each production batch (LOT) of the EXTRACTME PLASMID MIDI kits are tested using BLIRT’s ISO-certified quality management system. The purified DNA concentration, quality and stability are evaluated by gel electrophoresis and spectrophotometry. In addition, the functional quality is tested by qPCR, digestion with restriction enzymes and pDNA transfection.