UltraClean™ Tissue DNA Kit From Mo Bio Laboratories

The UltraClean™ Tissue DNA Kit is used for isolating pure genomic DNA from animal tissues and in vitro cultured cells. This kit has been tested for brain, lung, mouse-tails, liver, pancreas, heart, spleen, gallbladder, and bone marrow. I have used this kit for both cultured cells and mouse skin tissue. This kit comes in a small package for 50 preparations and large package for 250 preparations. DNA can be isolated in 30 minutes to 1 hour, depending upon the number of samples. First, tissue is homogenized with the help of innovative bead-beating technology; lysed cells are then applied to a spin column containing a silica membrane. The silica spin filter format eliminates the need to use any harmful organic solvents. DNA binds to the silica membrane, but enzyme inhibitors and other contaminants are washed away. The DNA is then eluted resulting in pure DNA up to 50 kb in length. The isolated DNA can be used for running PCR and other downstream applications.

Initially, we have to start with 1-25 mg of tissue or 5 million cultured cells to isolate DNA. Each filter has the binding capacity of 40 µg of DNA. Each kit contains three solutions: TD1, 2 and 3. TD1 is for cell lysis; TD2 is for washing away unwanted contaminants; TD3 is for elution of the DNA. The kit also contains Bead Solution Tubes, which is full of the beads for homogenization, and lysis solution for tissue samples (not required for cultured cells). The kit also includes spin filters in collection tubes for binding DNA and 2 ml microcentrifuge tubes for collecting the purified DNA. All of the solutions and accessories can also be purchased separately. This kit can be used for 1 year with efficient results.

For isolation of DNA from cultured cells solution, TD1 (approximately 500 µl) is directly added to the pellet of cells and then mixed well to disperse the pellet. After that, the solution is transferred to the spin filter and centrifuged at 10,000 g for 1 minute at room temperature. Solution TD 2 (400 µl) is then added and centrifuged again at the same speed. The resultant flow through is discarded. After this, we centrifuge again for 1 minute to eliminate any residual TD 2 wash solution. Finally, the spin filter is placed in the microcentrifuge tube and solution TD3 (10 mM Tris -50 µl) is added and centrifuged to elute the desired DNA. The DNA can be stored as such at -20ºC for further use. The spin filter can be discarded.

For tissue samples, we first have to homogenize the tissue with the help of the beads and lysis solution. Once the tissue is placed in the Bead Solution Tube, the tube is placed horizontally on a vortex adapter (can be purchased from Mo Bio Laboratories) and vortexed carefully. This is the most crucial step as the tissue should be grinded well so that the cells can be separated from each other. The amount of time this step takes varies according to the number and type of your tissue samples. Soft tissue can be homogenized in 5 minutes; other tissues can take more time. After homogenization, the tube is centrifuged at room temperature to get the supernatant. The supernatant is carefully removed and lysis solution equal to the volume of supernatant is added and the tube is vortexed again. After that, the steps are same as in the case of cultured cells (i.e. addition of Solutions TD2 and TD3). Sometimes it happens that we don’t get a clear solution after centrifuging the samples. This shows that either bead beating was not done efficiently and requires more time, or that we have taken too much tissue. Sometimes it happens that we don’t get high yield of DNA; this may be due to improper lysis of cells or overloading the columns.

My research aims to identify dietary constituents involved in cancer chemoprevention in both in vivo and in vitro models. Earlier, I was using a routine phenol-chloroform extraction method for DNA isolation from mouse skin tumor tissue. The DNA I used to get was not pure and because of that, I was not able to use it for PCR. Then I opted for this kit and got pure DNA from even the mouse skin which is the toughest one to get pure DNA from. Isolation of DNA from cultured cells is easier than that of isolation of DNA from animal tissue; it hardly takes 30 minutes to isolate DNA from cultured cells with this kit. The only precaution we have to take with animal tissue samples is that the lysed cells should be loaded in an amount appropriate to the binding capacity of the filter and lysis should be done properly.

The main advantage of this kit is that we don’t have to extract DNA using phenol-chloroform and the time consumed is much less that with traditional methods. I used the UltraClean-purified DNA for screening the early mutational changes occurring in mouse skin carcinogenesis induced by chemical carcinogen; I screened for these mutations using PCR. I was also able to identify apoptosis (via DNA fragmentation) as a hallmark of carcinogenesis induced by dietary constituents. We could also establish mutations in the E6/E7 oncogenes and the L1 gene of human papillomavirus 16 cervical cancers, from different stages of cervical carcinoma patients. DNA was isolated using this kit and samples were amplified using primers specific for the E6, E7 and partial L1 regions.

I would, therefore, recommend UltraClean™ Tissue DNA Kit for quick and reliable isolation of genomic DNA.