Dustin Edwards

Lab protocols

These are the lab protocols used in Dustin Edwards's lab at Tarleton State University, in two groups: phage protocols from the SEA-PHAGES course, and PCR primers with their reaction and cycling conditions. Each protocol has its own page with the full method. They are written for students in the lab and for anyone running the same methods elsewhere.

Phage methods#

  • Phage discovery guide: the Phage Discovery Program at Tarleton, with a link to the official SEA-PHAGES Phage Discovery Guide, which covers each stage from soil sample to annotated genome, and the places where the lab's practice differs from it.
  • Phage isolation protocol: isolating bacteriophages from soil and water by direct and enriched isolation, then plaque purification, spot and full plate titers, webbed plates and a high-titer phage lysate. It includes worked titer arithmetic and troubleshooting from several years of lab notebooks.
  • Phage DNA extraction protocol: the column-free ZnCl2/TES method the lab has used since 2018 to extract phage DNA from a high-titer lysate, with a note at each step on why that reagent is used. It ends with checks of DNA quantity and quality before sequencing and a rescue for salty or dilute DNA.

PCR and primers#

  • COI primers: LCO1490 and HCO2198: the Folmer primer sequences for COI barcoding PCR of invertebrate mitochondrial DNA, with the materials, the cycling table and the expected 708 bp product.
  • REV PCR primers: three primer sets used to detect reticuloendotheliosis virus (REV) in avian DNA, one in the 3′ LTR (8000-8297) and two in the pol gene (2500-3075 and 4777-5575), with sequences, cycling tables and product sizes. The research behind them is on the avian retroviruses page.
  • Pan-avian GAPDH PCR: primer sequences and PCR conditions for a 534 bp avian GAPDH product, used as a control that a bird DNA extraction holds DNA that will amplify, so a negative REV result can be trusted.

Retired protocols#

The older Wolbachia teaching protocols have been retired and are no longer published here. The COI barcoding primers used alongside them remain on the COI primers page.