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The EXTRACTME PLASMID DNA kit is designed for the rapid and efficient purification of high quality plasmid DNA from recombinant Escherichia coli strains. The isolation protocol and buffer formulations were optimized for high isolation efficiency and  DNA purity. The product is intended for research use only.
Bacterial broth culture
approx. 25 μg pDNA
approx. 25 minutes
A260/A280 ratio = 1.7 – 1.9
Isolation from high copy number plasmids
(such as pUC, pBluescript®, pGEM®, pJET, pTZ and derivatives)
For DNA isolation from high copy number plasmids, process pellets from 0.5-5 ml of bacterial culture. Do not exceed 5 ml, as this may result in clogging of the purification minicolumn and no increase in DNA yield will be obtained.
Isolation from medium and low copy number plasmids
(such as pBR322, pET, pACYC, pSC101 and derivatives, cosmids)
For DNA isolation from medium and low copy number plasmids, it may be necessary to increase the sample volume by as much as up to 10 ml in order to recover a sufficient quantity of DNA. The volumes of the buffers used for isolation should not be altered.
The DNA purification procedure utilises spin minicolumns with membranes which efficiently and selectively bind nucleic acids. In the first isolation step, the plasmid DNA 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 removed in the centrifugation step. The lysate is applied to the purification column membrane and the DNA is bound. The two-step washing stage effectively removes impurities and enzyme inhibitors. The purified plasmid DNA is eluted using a low ionic strength buffer (Elution Buffer) or water (pH 7.0-9.0) and may be used directly in all downstream applications such as PCR, qPCR, Southern blotting, DNA sequencing, enzymatic restriction, ligation and so forth, or stored until ready to use.
Quality control
The quality of each production batch (LOT) of the EXTRACTME PLASMID DNA kit is tested using standard QC procedures. The purified DNA concentration, quality and stability are evaluated by gel electrophoresis and spectrophotometer. In addition, the functional quality is tested by qPCR and digestion with restriction enzymes.