From specimen to sequence: Huang MEGA Lab Nanopore workflow

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Culture media

  1. Fungal culture media (LIMS login required)

Strain phenotyping

  1. Antifungal MIC (broth microdilution) and biofilm assays (crystal violet, MTT) (LIMS login required)

Molecular methods

  1. DNA extraction v9.0 (modified BOMB) (LIMS login required)
  2. Soil DNA extraction — semi-automated (SuperSoil, Zixpress 32) (LIMS login required)
  3. Soil DNA extraction — manual (SuperSoil, legacy) (LIMS login required)
  4. 2-step PCR w. barcoded-head primer pairs (environmental and mixed templates) (LIMS login required)
  5. One-pot PCR w. barcoded-head primers (strains: single- or multi-locus) (LIMS login required)
  6. Purification (size selection, 1X ratio) (LIMS login required)

Nanopore procedures

  1. Library construction (R10 NB barcode) (KAPA EvoPrep NBD114.96) (LIMS login required)
  2. Library construction (R10 PCR barcode) (KAPA HyperPrep) (LIMS login required)
  3. Flow cell wash (PromethION) (LIMS login required)

Miscellaneous

  1. RNAlater (sample preservation solution) recipe (LIMS login required)
  2. DEPC-treated water (nuclease-free water) (LIMS login required)