PRINCIPLE

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.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

Bacterial broth culture:
50-150 ml (high-copy number plasmids)
100-300 ml (medium- and low-copy number  plasmids)

YIELD

200-600 μg of transfection-grade pDNA from 100 ml of cultured bacterial cells

TIME REQUIRED

• 70 min for EM16 and 100 min for EM17
• additional ~60 min for DNA precipitation

DNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.7 – 1.9

ENDOTOXIN REMOVAL (EM17)

<0.1 EU/μg verified by LAL

RECOMMENDATIONS AND IMPORTANT NOTES