Gentra Puregene Kits enable purification of high-molecular-weight (100–200 kb) DNA suitable for archiving. The scalable purification procedure gently removes contaminants and inhibitors and allows samples to be purified for use as long-term references.
The purity of DNA has a significant effect on the accuracy of results obtained in downstream applications. Sensitive downstream applications, such as PCR, demand use of DNA of the highest quality and molecular weight for reliable results. Gentra Puregene Kits remove 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 figures " Archive-quality DNA", “ High-molecular-weight DNA from tissue” and “ High-molecular-weight DNA from mouse tails”) and performs well in sensitive downstream applications including PCR and restriction digestion (see figure " Efficient restriction endonuclease digestion").
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 Gentra Puregene Kits is highly stable and suited for use in a wide range of applications, such as:
Comparison of Gentra Puregene Kits
| Features | Gentra Puregene Tissue Kit | Gentra Puregene Buccal Cell Kit | Gentra Puregene Cell Kit | Gentra Puregene Blood Kit | Gentra Puregene Mouse Tail Kit | Gentra Puregene Yeast/Bact. Kit |
|---|---|---|---|---|---|---|
| Applications | PCR, restriction digest, Southern analysis, SNP analysis | PCR, restriction digest, Southern analysis, SNP analysis | PCR, restriction digest, Southern analysis, SNP analysis | PCR, restriction digest, Southern analysis, SNP analysis | PCR, restriction digest, Southern analysis, SNP analysis | PCR, restriction digest, Southern analysis, SNP analysis |
| Elution volume | 50–250 µl (varies) | 20 µl (varies) | 50–250 µl (varies) | 250 µl – 1000 µl | 50 µl (varies) | 50 µl (varies) |
| Format | Scalable | Scalable | Scalable | Scalable | Scalable | Scalable |
| Main sample type | Tissue samples, fixed and paraffin-embedded tissue | Buccal brush, mouth wash | Cultured cells | Whole blood, buffy coat, body fluid | Mouse tail | Gram+ and Gram- bacteria, yeast |
| Processing | Manual | Manual | Manual | Manual | Manual | Manual |
| Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein | Genomic DNA | Genomic DNA | Genomic DNA | Genomic DNA | Genomic DNA | Bacterial DNA / yeast DNA |
| Sample amount | 5–100 mg | 1 swab / 10 ml | 1 x 106 – 2.2 x 107 | 300 µl – 10 ml / 1 x 107 / 50 µl – 1000 µl | 5–10 mg | 0.5–1.5 x 109 cells / 1–2 x 108 cells |
| Technology | Modified salting-out precipitation method | Modified salting-out precipitation method | Modified salting-out precipitation method | Modified salting-out precipitation method | Modified salting-out precipitation method | Modified salting-out precipitation method |
| Time per run or per prep | 25–60 minutes (+ cell lysis + DNA rehydration) | 25–60 minutes (+ cell lysis + DNA rehydration) | 25–60 minutes (+ DNA rehydration) | 25–60 minutes (+ DNA rehydration) | 25–60 minutes (+ cell lysis + DNA rehydration) | 25–60 minutes (+ DNA rehydration) |
| Yield | Varies | 1 µg/swab / 46 µg/10 ml mouth wash | 7 µg / 1 million cells | 35 µg / ml whole blood | 10–75 µg | 17–50 µg/ml culture / 4.5 µg/ml culture |
