Paul Burr, Wellcome Clinical Scholar, Department of Veterinary Pathology, University of Glasgow, Bearsden Rd, Glasgow. G61 1QH.
A method that can be conveniently used to prepare DNA from a wide range of tissues for analysis in the Polymerase Chain Reaction is described. The method is both quick and simple, with a low risk of inadvertent contamination of samples due to the reduced number of manipulations required compared to other DNA extraction methods.
Canine tissues (tri-geminal ganglion, lumbo-sacral ganglion, brain, mesenteric lymph node, sub-mandibular lymph node, tonsil, sub-mandibular salivary gland, parotid salivary gland, spleen, liver and kidney) were collected from a number of dogs during routine post mortem. All tissues were collected within 2 hours of death and frozen at -70 C until processed. Great care was taken during preparation to avoid problems of both environmental and cross contamination.
1. Cut a small (3 mm3) piece of tissue on a fresh glass slide using a fresh scalpel blade. Macerate slightly with the blade and place in a sterile 1.5 ml screw cap Eppendorf tube.
2. Add 1 ml of 1x Trypsin/EDTA (Gibco BRL). Incubate at room temperature for 15-30 minutes, shaking occasionally.
3. Spin down (13K, 2-3 minutes) to pellet tissue, and carefully remove all the supernatant.
4. Add 250 l of InstaGene matrix (Bio-Rad Laboratories), and mix well. Incubate at 56 C for 30 minutes, mixing occasionally.
5. Vortex tube thoroughly for 10 seconds, place in boiling water bath or 100 C heat block for 8 minutes.
6. Vortex thoroughly for 10 seconds, spin at 13K for 2-3 minutes.