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The two-step SYBR® Green RT-PCR was performed in a final volume of 10 L: 2.5 L of molecular biology grade water (Mo Bio Laboratories, Inc.), 1 L of 20 pg/µL of each primer (final concentration of 10 µM), 0.2 L of X12 kit reaction mixture, 1 L of 5 pg/µL of DNA for the test sample, and 1.7 L of RNAse-free water. The same reaction was performed for 5-fold serial dilutions of DNA extracted from blood of a healthy human mixed with DNA of FS free of infection from T.infestans to estimate the sensitivity of the X12-qPCR reaction. The thermal profile included 3 min of pre-incubation at 95°C and 40 amplification cycles (95°C for 5 sec, 60°C for 30 sec and 72°C for 30 sec). The measurement of emitted fluorescence was performed at 72°C at the end of each cycle. After all the amplification cycles a melting curve was run. The results were analyzed with the MxPro v4.1 d (Agilent Technologies) software.
The primers used for real-time RT-PCR amplification of PRRSV were designed using sequence data from the PRRSV N protein gene. The partial sequence of the N gene of PRRSV strain ch-1a was downloaded from GenBank (accession no.
It wasn't until 1989 that Sycamore had a real police department. The first suspect in the Ridulph case was arrested in '79. A lot of people blamed the cops. Maybe a lot of people blamed the girl. If the case had been solved four years earlier, the city of Sycamore might have gotten that precious $1 million in federal money that it had been waiting for. And the state might have gotten its share of the money, too.
He dumped Kathy, who was a good student and a model student. After years of playing to the crowd, he started his own band and wouldn't return her calls, though it took her a decade to track him down. Having tried his hand at everything, including selling pot in his senior year of high school, he was busted at age 18 for having sex with a 15-year-old girl. He was given a year in a work-release program, and then moved in with his mother, who paid his fines. 827ec27edc