Puregene DNA procedure.|Reproducible purification of archive-quality DNA.|
The simple Puregene procedure uses a modified salting-out precipitation method for purification of DNA. No mixing or dilution of solutions is necessary, and hands-on time is minimized. The procedure provides convenient stopping points that allow safe, temporary storage of partially purified samples.|DNA was purified from 8 replicate IM9 cultured cell samples (1.5 x 108 cells each) using the Gentra Puregene Cell Kit. Purified DNA was analyzed by agarose gel electrophoresis. M: Lambda HindIII marker.|
Purity of DNA has a significant effect on the accuracy of results obtained in downstream applications. Sensitive downstream applications, such as PCR, demand the use of DNA of the highest quality and molecular weight for reliable results. The Gentra Puregene Cell Kit removes contaminants and enzyme inhibitors enabling purification of highly stable DNA well-suited for archiving. Purified DNA is of high quality demonstrated by molecular weights of 100–200 kb (see figure "Reproducible purification of archive-quality DNA").
Cells are lysed with an anionic detergent in the presence of a DNA stabilizer. The DNA stabilizer limits the activity of intracellular DNases and also DNases found elsewhere in the environment. RNA is then removed by treatment with an RNA digesting enzyme. Other contaminants, such as proteins, are removed by salt precipitation. Finally, the genomic DNA is recovered by precipitation with alcohol and dissolved in hydration solution (1 mM EDTA, 10 mM Tris·Cl pH 7.5). Purified DNA typically has an A260/A280 ratio between 1.7 and 1.9, and is up to 200 kb in size. The DNA can be safely stored at 2–8°C, –20°C, or –80°C.
The simple Puregene procedure uses a modified salting-out precipitation method for purification of DNA (see flowchart "Puregene DNA procedure
"). No mixing or dilution of solutions is necessary, and hands-on time is minimized. The procedure provides convenient stopping points that allow safe, temporary storage of partially purified samples.
DNA purified using the Gentra Puregene Cell Kit is highly stable and suited for use in a wide range of applications, such as:
- DNA archiving
- SNP analysis
- Southern blotting