Normal brain homogenates (NBH) from either human PrP (M129 allele, tg650 line) [24 (link)] or ovine PrP (VRQ allele, tg338 line) [22 (link)] transgenic mice that over express PrPC, 6 and 8 times respectively, were used as substrates for PMCA. After collection, mouse brains were rinsed in cold PBS and immediately frozen on dry ice before long-term storage at -80°C. Brains were then homogenized at a 10% (w/v) concentration in a conversion buffer composed of 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail (Roche) in PBS (pH 7.2). Homogenates were clarified at 2000 x g for 20 seconds and frozen at -80°C in single-experiment aliquots.
Wires were inserted into 0.2 ml PCR-tubes and mixed with 90 μl of 7.5% NBH in conversion buffer for Surf-PMCA amplification (Misonix 4000, N.Y., USA). One PMCA round was composed of 80 cycles of 20 seconds of sonication at 220–240 W power followed by 29 min 40 seconds of incubation at 37°C. Ten μl of amplified product was mixed with 90 μl of fresh NBH in conversion buffer and subjected to an additional PMCA round of 80 cycles. Three or four rounds were performed depending on the prion strain.
Wires were inserted into 0.2 ml PCR-tubes and mixed with 90 μl of 7.5% NBH in conversion buffer for Surf-PMCA amplification (Misonix 4000, N.Y., USA). One PMCA round was composed of 80 cycles of 20 seconds of sonication at 220–240 W power followed by 29 min 40 seconds of incubation at 37°C. Ten μl of amplified product was mixed with 90 μl of fresh NBH in conversion buffer and subjected to an additional PMCA round of 80 cycles. Three or four rounds were performed depending on the prion strain.
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