24,00 € – 235,00 €EXTRACTME PLASMID MINI KIT is designed for a 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. For mini-scale extraction of plasmid DNA from recombinant Escherichia coli strains; binding capacity 25 μg pDNA; isolation time: about 25 minutes.
DNA purification procedure utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. During the first step plasmid DNA is released from bacterial cells by alkaline lysis. Then lysate is neutralized and all cell residues along with proteins and genomic DNA are separated in centrifugation step. Lysate is applied to purification minicolumn membrane and DNA is bound.
A two-step washing stage effectively removes impurities and enzyme inhibitors. Purified plasmid DNA is eluted with then use of 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.
Bacterial broth culture, frozen cell pellet – however the efficiency will be decreased
Approx. 60 μg DNA
Approx. 25 minutes
A260/A280 ratio = 1.7 – 1.9
RECOMMENDATIONS AND IMPORTANT NOTES
Sample material DNA isolation efficiency and purity can vary depending on a number of factors, such as plasmid copy number (high copy and low copy plasmids), cell density of bacterial culture, cell type (morphology), culture medium, growth phase, age and condition of cells. It is recommended to extract DNA from fresh, properly prepared starting material, which guarantees best isolation parameters.
To minimize DNA degradation, avoid subjecting the sample material to repeated freeze/thaw cycles. Using old or repeatedly frozen/thawed material may result in low efficiency of isolation and poor DNA quality.
In the table below shows the optimal culture volume that should be used for isolation according to the optical density at 600 nm.
EXTRACTME PLASMID MINI KIT – TROUBLESHOOTING
BalticBlue is an indicatior that provides visual identification of optimum buffer mixing. This prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA and cell debris.
BalticBlue should be added to Resuspension Buffer at a ratio of 1:1000 (e.g. 12.5 μl BalticBlue to 12.5 ml Resuspension Buffer). Mix thoroughly. BalticBlue precipitates. After addition of Lysis Buffer, precipitate dissolves and color changes to blue. During neutralization invert mixture gently until white precipitate is formed and blue solution will be colorless.
When isolating in parts, transfer enough of Resuspension Buffer for your isolations to a separate bottle/tube and add BalticBlue.
The quality of each production batch (LOT) of the EXTRACTME PLASMID MINI KIT is tested using standard QC procedures. Purified DNA concentration and quality are evaluated by gel electrophoresis and spectrophotometer.