The EXTRACTME PLASMID MAXI ENDOTOXIN-FREE kit (EM19) and EXTRACTME PLASMID MAXI (EM18) kit are designed for the efficient purification of high quality plasmid DNA from 200-1000 ml of cultured bacterial cells. The kits are based on anion-exchange resins, allowing extraction of ultrapure, transfectiongrade 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.
|EM19-025||25 extractions||Ask for an offer|
|EM19-010||10 extractions||Ask for an offer|
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:
200-600 ml (high-copy number plasmids)
400-1000 ml (medium- and low-copy number plasmids)
1.0-1.5 mg of transfection-grade pDNA from 400 ml of cultured bacterial cells
- 90 min for EM18 and 120 min for EM19
- 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
EXTRACTME PLASMID MAXI ENDOTOXIN-FREE – TROUBLESHOOTING
The quality of each production batch (LOT) of the EXTRACTME PLASMID MAXI 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.