May 04, 2026
Version 5
Modified Promega Wizard Extraction for Barcoding Macrofungi V.5
- 1Mycota Lab / Biodiverse / MycoMap;
- 2Mycota Lab
- Mycota Lab
Protocol Citation: Stephen Douglas Russell, Ryan Peace 2026. Modified Promega Wizard Extraction for Barcoding Macrofungi. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb3p4vx1/v5Version created by Stephen D Douglas Russell
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 04, 2026
Last Modified: May 04, 2026
Protocol Integer ID: 316300
Keywords: preparing macrofungal specimen, macrofungal specimens for sanger, secondary extraction protocol for ont nanopore, modified promega wizard extraction for barcoding macrofungi, general fungal collection, dna extraction, nanopore barcoding, dna extraction method, result with nanopore barcoding, fungi, polypore, older museum collections of macrofungi, modified promega wizard extraction, quick extraction protocol, ont nanopore, extraction protocol, extraction, barcoding macrofungi, primary extraction protocol, important specimen, secondary extraction protocol, macrofungi, recalcitrant species, nanopore, clean dna, more specimen, sequencing, older museum collection, dna
Abstract
This protocol is best used when preparing macrofungal specimens for Sanger sequencing or as a secondary extraction protocol for ONT nanopore barcoding. We also utilize it as a primary extraction protocol for working with older museum collections of macrofungi when clean DNA is needed to be retained with the collections.
Each step in this protocol can be expanded to work with much larger sets. It is common for us to work with 96 or more specimens at a time.
Materials
Equipment:
Tube Racks for 1.5uL eppi tubes
Tweezers
Pestles
Heat Block
Vortexer
Centrifuge
Consumables:
1.5uL eppi tubes
Molecular water
70% ethanol
Kimwipes
Reagents:
Nuclei Lysis Solution, 1000mlPromegaCatalog #A7943
Protein Precipitation Solution 350mlPromegaCatalog #A7953
IsopropanolIBI Scientific
Protocol materials
Nuclei Lysis Solution, 1000mlPromegaCatalog #A7943
Protein Precipitation Solution 350mlPromegaCatalog #A7953
IsopropanolIBI Scientific
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 500uL of Nuclei Lysis Solution, 1000mlPromegaCatalog #A7943 to 1.5mL eppi tubes containing your tissue.
Grind the tissue well in each tube. Historically we used a sterile pestle to manually grind the tissue. This does not scale very well. Currently we place a 2mm sterile stainless steel bead/bearing (from Amazon) into the microcentrifuge tube and vortex it on an Onmi BeadRuptor Elite. 90 seconds on, 10 seconds off, 90 seconds on. Speed: 3.55.
Note: For herbarium specimens the Omni cycles assume the tissue was preground at the herbarium with tweezers during sampling.
Heat the tubes at 80 °C for 00:10:00 .
10m
Centrifuge the tubes for 00:03:00 at max rpm. You can start to setup your PPS tubes from Step 6 while this is finishing.
3m
Add 200 µL of Protein Precipitation Solution 350mlPromegaCatalog #A7953 to a new 1.5mL microcentrifuge tube. Label your tube.
Note: 100 µL can frequently work as well as 200 here, and it will take this expensive reagent twice as far. Might be good to test with your sample types if you have many to do.
Transfer the supernatant (liquid on top) to the new tube using a 1000uL pipette.
Vortex the tube for 00:00:20 .
Centrifuge the tube for 00:06:00 at max rpm. You can start to setup your Iso tubes from Step 7 while this is finishing.
Note: At this point the supernatant above the pellet should not be cloudy. It is fine for it to be colored, as long as it is clear. If the liquid above the pellet is cloudy, it is best to repeat this step to continue cleaning it before moving on to the next step - put the partially cleaned supernatant (fluid above the pellet) into a new tube, 200 PPS, vortex again, centrifuge again, and it should be clearer.
6m 20s
Add 500 µL of 100%IsopropanolIBI Scientific to a new 1.5mL microcentrifuge tube. Label your tubes.
Transfer the supernatant (liquid on top) to the new tube. It can just be poured out of the old tube into the new one directly.
^^^ Above this point: DNA is in the liquid solution/supernatant. ^^^
-------------------------
⌄⌄⌄ Below this point: DNA is precipitated and will be attached to the bottom of the tube. ⌄⌄⌄
Centrifuge the tube for 00:01:00 . 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. No need to pipette.
1m
Add 500 µL of 70% ethanol to the tube.
Centrifuge the tube for 00:01:00 .
Discard the supernatant. It can just be poured directly out of the tube into a waste container. No need to pipette.
Place the tube upside down on a Kimwipe for at least 00:15:00 , or until all of the ethanol has evaporated from the tube. I usually leave the tube to dry overnight. This does not have to be in a sterile environment. We often use a small box, plastic bin, or drawer.
16m
Add 30uL of molecular water to the tube.
Your DNA template is now ready for amplification.