Modified Promega Wizard Extraction for Barcoding Macrofungi
Stephen Douglas Russell
Abstract
'This protocol is best used when preparing macrofungal specimens for Sanger sequencing or as a secondary extraction protocol for ONT nanopore barcoding.
The quality of a DNA extraction method is a primary limiting factor in the total number of samples that will return a result with nanopore barcoding of fungi. The "quick" extraction protocol will often yield a positive result for 80-85% of general fungal collections (less if biased with polypores and recalcitrant species). Utilizing this extraction protocol pushes that number to nearly 100%. It is more time consuming and utilizes more expensive chemicals, but may be worth considering for important specimens that fail with the quick extraction protocol.
Steps
Place tissue from your specimens into each tube using tweezers. Utilize a piece about the size of a grain of rice or smaller. It should easily drop to the bottom of the tube. The tissue can be either fresh or dried. Label the tube with the appropriate number. Wipe the tweezers off with a Kimwipe or paper towel in between each specimen. These tubes can be stored at room temperature until they are ready to be used.
Add 600uL of
Grind the tissue well in each tube using a sterile pestle.
Heat the tubes at 65°C for at least 0h 15m 0s. It is fine to leave it in longer. I often use one hour.
Centrifuge the tubes for 0h 3m 0s at max rpm.
Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. Label your tubes.
Add 200µL of
Vortex the tube for 0h 0m 20s.
Centrifuge the tube for 0h 6m 0s at max rpm.
Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. Label your tubes
Add 600µL of 100%
Centrifuge the tube for 0h 1m 0s. The DNA will now be in a pellet stuck to the bottom of the tube.
Discard the supernatant. It can just be poured out of the tube into a waste container.
Add 600µL of 70% ethanol to the tube.
Centrifuge the tube for 0h 1m 0s.
Discard the supernatant. It can just be poured directly out of the tube into a waste container.
Place the tube upside down on a Kimwipe for at least 0h 15m 0s, or until all of the ethanol has evaporated from the tube. I usually leave the tube to dry overnight.
Add 30uL of molecular water to the tube.
Your DNA template is now ready for amplification.
