title: Pyrosequencing read of marine bioaerosol sample: uncultured bacteria: Sample H24
design: "Due to the small yield of PCR products with the pyrosequencing tagged primer, a reamplification PCR for the prokaryotic 16S gene was conducted. In a first PCR reaction, the eubacterial forward primer GM3 and the reverse primer 907 (Muyzer et al. 1995) were applied with each 5 ng DNA as template. Subsequently, 1 µl of the reaction was used for a PCR with pyrosequencing tagged primers (Krause et al. 2012). PCR reactions were run with 2.5 µl taq buffer (10x), 5 µl TaqMaster PCR Enhancer (5x), 0.7 µl of each primer (20 mM), 0.75 µl dNTPs (2.5 mM each), and 3 U of taq DNA polymerase (5 Prime, Hamburg, Germany), and were adjusted with ultrapure water to a reaction volume of 25 ul. The conditions for the PCR were the following: 94 °C for 10 min, followed by 30 cycles with 94 °C for 1 min, 44 °C for 1:30 min and 68 °C for 2 min; final elongation at 68 °C for 5 min. PCR products were then verified by gel electrophoreses and by the absence of amplifications in DNA-free controls. PCR products were purified with the peqGOLD Gel Extraction Kit (peqlab, Erlangen, Germany). Sequencing was done on a Roche 454 GS-FLX Titanium platform (LGC Genomics, Berlin, Germany)."