Fusarium EF1alpha mock community: experiment 2 nested mix 1 replicate 3: Sample Fusarium10_nested
"Amplicons of the Fusarium transcription elongation factor (EF-1?) were generated from mock communities and field samples. Fusarium mock communities consisting of DNA from ten species were prepared in three replicates and run in three individually tagged PCR replicates. Field samples were from soil and wheat kernels. Samples were run in either a nested PCR (mock communities, soil samples) with Fa+7/Ra+6 and Fa/Ra primers or a single PCR (mock communities, kernel samples) with only Fa/Ra (Edel-Hermann et al 2015, DOI: 10.1016/j.mimet.2015.01.026). Both PCRs were run in 25 µl reactions with 0.8 ng/µl template or 2 µl PCR product, 190 µM of each nucleotide, 2.75 mM MgCl2, primers at 200 nM and 0.12 U/µl polymerase (DreamTaq Green, Thermo Scientific, MA, USA) in PCR buffer. PCR conditions were 3 min at 94°C, 20 cycles (for Fa+7/Ra+6) and 25 or a variable number of cycles (for Fa/Ra) of [1 min at 94°C, 1 min at 67°C, 1 min at 72°C] and 10 min at 72°C. The Fa primer was tagged with a 9 bp sample identifying tag. The number of cycles of the Fa/Ra assay was adapted for each field sample ranging 20-35. PCR products were cleaned using AMPure (Beckman Coulter, CA, USA). DNA concentration was measured using capillary electrophoresis on a MultiNA (Shimadzu, Japan). The sample pool was sent to Eurofins MWG Gmbh (Ebersberg, Germany) for adaptor ligation and sequencing on 1/4th of a plate on a GS FLX++ 454 sequencer (Roche, Switzerland). Before sequencing the amplicon pool was purified by gel extraction. "