Ez 1 dna extraction robot

DNA from blood samples was extracted using the EZ-1 DNA extraction robot (Qiagen) from 200l of blood after one hour incubation at 55°C to inactivate virus. To recover DNA from swabs (oral, nasal, rectal, scarification site lesion), 400l of PBS was added to each swab and the swab extraction tube systems (SETS; Roche) protocol was followed to recover a homogenate. DNA from swab samples was obtained from 100l of the swab homogenate (after the homogenate was incubated with Proteinase K and Buffer G2 at 55°C to inactivate virus) using EZ-1 DNA extraction robot (Qiagen); the remaining swab homogenate was used for virus isolation (see below). Tissue samples were placed in disposable dounce homogenizers with 1 ml of PBS and ground thoroughly to create a slurry. Genomic DNA was obtained form 100l of tissue slurry (after the slurry was incubated with Proteinase K and Buffer G2 at 55°C to inactivate virus) with EZ-1 DNA extraction robot (Qiagen) and the remaining slurries were used for virus isolation (see below). All sample processing and testing was performed under BSL-2 conditions with BSL-3 work practices.

DNA was extracted from the frozen fecal samples using an extraction method previously described (16 (link)). Briefly, 50 mg of the samples were mixed with 1 ml of sterile PBS buffer and incubated for 10 min prior to a bead beating on an Eppendorf Mixer (model 5432, Eppendorf, Hamburg, Germany) at 4°C for 30 min. The samples were centrifuged at 13,000 rpm for 1 min and about, 200 ml of the supernatant was transferred to a clean 2.0 ml sample tube. DNA was extracted from the supernatant using the EZ1 DNA extraction robot (Qiagen bioinformatics, Aarhus, Denmark) and the DNA tissue kit (Qiagen bioinformatics, Aarhus, Denmark). The extracted DNA was frozen at −20°C until further analysis.