The EXTRACTME PLASMID MIDI kit (EM16) and are designed for ultrapure, transfection-grade plasmid DNA isolation im medium scale (50-300 ml of bacterial culture); yield: 200-600 μg DNA from 100 ml culture; isolation time: 120-130 minutes (with DNA precipitation); centrifugation steps: 6000 x g (no need to have ultracentrifuge); laskyophilized RNase A included; each column contain special modificated anion-exchange resin.
The product is intended for research use only.
|EM16-025||25 extractions||Ask for an offer|
|EM16-010||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:
50-150 ml (high-copy number plasmids)
100-300 ml (medium- and low-copy number plasmids)
200-600 μg of transfection-grade pDNA from 100 ml of cultured bacterial cells
- 70 min for EM16 and 100 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
EXTRACTME PLASMID MIDI – 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.