Extraction of DNA from a single-parasite

from Matt Fisher's Thesis

Single S. ratti larvae or free-living adults were transferred from faecal cultures to 2 ml of distilled water to remove adhering debris. The worms were transferred into a microfuge tube in 5 microl of distilled water, together with 0.5 microl 10 X PCR buffer (Dynazyme) and 0.5 microl Proteinase K (20 mg ml^-1 ) (Boehringer Mannheim), then overlaid with a drop of mineral oil (Sigma). After centrifugation at 10 000 g for 3 minutes the sample was held at -70 ^oC for 30 minutes, 55 ^oC for 90 minutes and 95 ^oC for 30 minutes in a thermocycler (Biometra). The DNA preparations were stored at -20 ^oC.