The following protocols are developed for mycology research in the Research and Conservation Department at Denver Botanic Gardens, but may be used as general guidelines for similar research projects or programs.

This section is a work in progress. Please return periodically to find more protocols and instructions in everything from collecting specimens in the field, to DNA extraction and sequencing.

Field Protocols

• Denver Botanic Garden’s Field Collecting Protocol for Macrofungi – HERE
• Making fungal observations using iNaturalist for the Colorado Mycoflora Project

Herbarium Protocols

• Stains and Reagents for Mycology. By Ed Lubow.
• Using the Mycology Collections Portal (MycoPortal) to reference collections.
• Accessioning Specimens in the Sam Mitchel Herbarium of Fungi
• Else Vellinga’s Glossary of Macroscopic and Microscopic features of Macrofungi. From Flora Agaricina Neerlandica 1.

Laboratory Protocols

• DNA Extraction
  • Simple extraction – This is the “quick and dirty” method. Works well. Is cheap. However, is not intended for long-term DNA storage.
  • Omega Biotech: EZNA Fungal Miniprep “Cheatsheet” – We use this method to produce ‘clean’ DNA for longterm storage of quality DNA samples.
• PCR protocol (Sanger prep and most other PCR) – Our protocol uses the Bioline MyTaq mix.
  • Primer page. (Coming soon[ish])
• HT DNA Metabarcoding of Macrofungal Specimens – From Gary Olds’ thesis project. Pre-pub on BioRxiv. Peer reviewed in Applications In Plant Sciences (in review June 2022). All sequencing is performed through the GBRC at the Univerisity of Idaho.
  • Primers and sequencing adapters for Illumina MiSeq. PCR1. PCR2.
  • Template for tracking samples .xlsx.
  • Sample Sheet prep instructions. (Coming soon[ish])
  • Main Protocol PDF.
  • GBRC documents .zip file.
• Gel Electrophoresis – Pretty standard (Coming soon[ish])
  • Pouring Gels.
  • TBE buffer instructions.
• PCR Clean and DNA quantification (Sanger and most methods) – We use a simple two step alcohol precipitation to clean PCR product. For DNA quantification we have a NanoDrop (not recommended for precise measurements) and a Qubit.
  • Cycle sequencing clean – The Field Museum method. Basically an alcohol precip method but with EDTH buffer. (Coming soon[ish])
• Sanger Sequencing Shipping Instructions (Coming soon[ish])
• High Throughput Shipping Instructions (Coming soon[ish])

Data Analysis

• Editing ABI files in Geneious (Coming soon[ish])
• Assembling Datasets and Performing Phylogenetic Analyses – This process is pretty convoluted. It requires a number of steps that can easily get lost in translation in a document like this. Please reach out with questions if you have them.