The DNeasy Blood & Tissue Kit allows for fast and easy purification of total DNA from a variety of sample sources, including fresh or frozen animal and plant tissue or cells, blood, bacteria, yeast, or viruses. The purified DNA is free of inhibitors, proteins, nucleases and other contaminants. Therefore, the purified DNA can be used for a variety of downstream applications such as PCR, Southern hybridization, RAPD, AFLP, RFLP, sequencing and cloning. DNeasy purified DNA is up to 50 kb in size, with fragments of approximately 30 kb size predominating; very small DNA fragments of 100 bp are also recovered.
Samples are purified on a silica column. First, samples are lysed in the presence of proteinase K. The lysate buffering conditions are adjusted to allow optimal binding of the DNA to the DNeasy mini spin column membrane. The membrane selectively binds DNA while contaminants pass through. With the DNA bound to the column, the membrane is washed in two centrifugation steps. The use of two different wash buffers significantly improves the purity of the eluted DNA. The wash buffers ensure complete removal of any residual contaminants without affecting the DNA binding. DNA can be eluted with buffer or water. Purification requires no phenol-chloroform extraction or alcohol precipitation, and involves minimal handling. This makes the DNeasy Blood & Tissue Kit highly suited for simultaneous processing of multiple samples.
This kit is available in two different formats, based on throughput requirements. The low throughput system uses spin columns and takes 20-30 min after lysis (depending on sample number). For higher-throughput applications, the DNeasy 96 Blood & Tissue Kit enables simultaneous processing of 96 or 192 samples in just one hour after lysis. However, the DNeasy 96 Blood & Tissue Kit requires a special centrifugation system called the Qiagen 96-Well-Plate Centrifugation System which is comprised of a plate rotor and a table-top centrifuge (4-15C or 4K15C) which enables purification of DNA from up to 2 x 96 samples in parallel. Both formats of the DNeasy Blood & Tissue Kit are suitable for both very small (only 100 cells) and large sample sizes (upto 5 x 106 cells).
Personally, I used spin column based DNeasy Blood & Tissue Kit for DNA extraction from mouse blood, fresh and frozen animal tissue (spleen and liver), human (Mo7e) and murine (Baf3) cell lines. Purified DNA was used for various downstream applications such as PCR amplification, restriction digestion as well as southern hybridization. I have had good success with this kit. The instructions are very easy to follow and all the necessary reagents are provided with the kit. The only special equipment required is a small tabletop centrifuge or vacuum manifold. In order to obtain optimum DNA yield and quality, it is highly recommended to not overload the DNeasy spin column. Therefore, I always used the recommended sample volume. Secondly, lysis time varies depending on the type of tissue processed. Lysis is usually complete in 1–3 h but sometimes frozen animal tissue samples were difficult to lyse. I usually left these samples overnight at 56ºC in ATL Buffer (provided) for efficient lysis. Sometimes you might need mechanical disruption of tissue which might reduce lysis time though overnight lysis works very efficiently and does not adversely affect DNA integrity or yield.
The DNeasy Blood & Tissue Kits copurify DNA and RNA when both are present in the sample. RNA does not affect PCR, but may inhibit other downstream enzymatic reactions. If RNA-free genomic DNA is required, RNase A (not provided with the kit) should be added to the sample before addition of Buffer AL, to digest the RNA.
Overall, this kit is easy to use, fast, very straightforward and relatively affordable. The kits can be purchased in quantities of 50 or 250 columns. I have had great success with this kit and highly recommend it.
Post-Doctoral Fellow
Oregon Health Sciences University