Analytical Biochemistry 348 (2006) 160–162 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY 0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.10.004 Notes & Tips Isolation of high-quality DNA for genotyping from feces of rodents Chris Murgatroyd a,¤ , Denys Bilko b , Dietmar Spengler a a Max-Planck Institute of Psychiatry, 80804 Munich, Germany b Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK Received 29 August 2005 Available online 24 October 2005 We present a simpliWed DNA extraction protocol using rodent feces allowing the isolation of pure and suYcient DNA suitable for analysis in genotyping studies. This pro- cedure is an easy, practical, and reliable alternative to the stressful current procedures of tail clipping and ear notch- ing in rats and mice, and it appears to be of particular inter- est in cases where animals are studied for behavior and stress responses. The most widely employed protocols for isolating rodent DNA entail tail clipping, ear notching, and toe amputation, all of which yield high concentrations of DNA [1–3]. The sixth report of the British Veterinary Association Animal Welfare Foundation/Fund for the Replacement of Animals in Medical Experiments/Royal Society for the Prevention of Cruelty to Animals/Universities Federation for Animal Welfare (BVAAWF/FRAME/RSPCA/UFAW) 1 Joint Working Group on ReWnement recommends that tail biop- sies be taken from mice between 3 and 4 weeks of age [4]. It further suggests that some kind of analgesia be used because mice and rats more than 4 weeks of age have fully ossiWed tails, increasing the risk of greater trauma. Although the procedure of tail biopsies has been routinely implicated in the Weld of biology since the early 1980s, the impacts of tail biopsy on the behavior, health, and mobility of rodents are still largely unknown. Even at a young age, however, some components of the sensory nervous system are fully intact in rodents and perception of noxious stimuli may occur to some degree [5]. Moreover, neonatal pain or trauma, but not necessarily a single acute episode, can inXu- ence subsequent development and behavior [6–9]. Therefore, the use of noninvasive sources of DNA in rodent studies is an attractive alternative, and previous approaches have detailed the feasibility of hair [10] and saliva [11] as sources in PCR-based experiments. Subse- quent work to obtain DNA from feces of nonrodents has described the procedure as unreliable, labor intensive, and requiring replications due to the poor quality of the sam- ples and the presence of bacterial DNA and PCR inhibitors [12–14]. Although one study has succeeded in purifying DNA from mice whole feces pellets using spin column chromatography [15], we present here an improved and simpliWed approach that we believe is more reliable and cost-eVective. Fresh feces were collected from C57BL6 mice and Wistar rats. Rodent epithelia regenerate every 3 to 4 days [16], sloughing the old cells into the lumen, where they then adhere to the fecal pellet during passage. Therefore, we postulated that the outermost fecal material may be covered with rather fresh intact cells from the distal colon and rectum suVering from less DNA degradation than those cells incorporated at earlier stages into deeper layers of the pellet, as has been supported in previous studies [12,17]. For each extraction, a single fecal pellet was brieXy washed with 0.001% SDS in an Eppendorf tube and was vortexed for 5 min to allow the detergent to dis- rupt the membranes of eukaryotic cells releasing the DNA. The sample was then centrifuged for 10 min in a benchtop centrifuge (14,000g at 4 °C) to separate cellular debris and undigested food from the sample. The superna- tant was removed and extracted by phenol, phenol/chlo- roform (1:1), and chloroform. The aqueous phase was precipitated by the addition of 10 l of 3 M sodium ace- tate (pH 5.2), 1 l of 1% glycogen, and 200 l of 100% eth- anol. Samples were placed either in liquid nitrogen for 5 min or at ¡20 °C overnight, and DNA was collected by * Corresponding author. E-mail address: murgatroyd@mpipsykl.mpg.de (C. Murgatroyd). 1 Abbreviations used: BVAAWF/FRAME/RSPCA/UFAW, British Vet- erinary Association Animal Welfare Foundation/Fund for the Replace- ment of Animals in Medical Experiments/Royal Society for the Prevention of Cruelty to Animals/Universities Federation for Animal Welfare; dNTP, deoxyribonucleoside triphosphate.