Author: Zhang, Chi; Xiu, Leshan; Xiao, Yan; Xie, Zhengde; Ren, Lili; Peng, Junping
Title: Simultaneous Detection of Key Bacterial Pathogens Related to Pneumonia and Meningitis Using Multiplex PCR Coupled With Mass Spectrometry Document date: 2018_4_5
ID: 0k2blp7n_11
Snippet: Twelve pairs of PCR primers were pooled and then mixed with hot start PCR enzyme, PCR buffer, MgCl 2 (Agena Bioscience, Inc.), uracil-DNA glycosylase (ShineGene Molecular Biotechnology, Shanghai, China), dNTP (dATP, dGTP, dCTP, and dUTP) (Promega, Madison, WI, USA), and 2 µl of DNA template for a 5-µl total volume. The multiplex PCR was performed in 384-well PCR plates using a ProFlex PCR system (Applied Biosciences, Foster City, CA, USA). The .....
Document: Twelve pairs of PCR primers were pooled and then mixed with hot start PCR enzyme, PCR buffer, MgCl 2 (Agena Bioscience, Inc.), uracil-DNA glycosylase (ShineGene Molecular Biotechnology, Shanghai, China), dNTP (dATP, dGTP, dCTP, and dUTP) (Promega, Madison, WI, USA), and 2 µl of DNA template for a 5-µl total volume. The multiplex PCR was performed in 384-well PCR plates using a ProFlex PCR system (Applied Biosciences, Foster City, CA, USA). The reaction conditions for the multiplex PCR were as follows: 45 • C for 2 min; 94 • C for 4 min; 95 • C for 2 min; 45 cycles of 95 • C for 30 s, 56.5 • C for 30 s, and 72 • C for 1 min; and 72 • C for 5 min. After amplification, shrimp alkaline phosphatase (SAP) (Agena Bioscience, Inc.) was used to dephosphorylate excess dNTP at 37 • C for 40 min, and then the SAP was inactivated at 85 • C for 5 min.
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