Mar 03, 2024
Version 3
Modified Promega Wizard Extraction for Barcoding Macrofungi V.3
- 1Mycota Lab / The Hoosier Mushroom Society
Protocol Citation: Stephen Douglas Russell 2024. Modified Promega Wizard Extraction for Barcoding Macrofungi. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb3p4vx1/v3Version created by Stephen 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: March 03, 2024
Last Modified: March 03, 2024
Protocol Integer ID: 96058
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.
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
Step 2
Protein Precipitation Solution 350mlPromegaCatalog #A7953
Step 6
IsopropanolIBI Scientific
Step 7
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 Nuclei Lysis Solution, 1000mlPromegaCatalog #A7943 to 1.5mL eppi tubes containing your tissue.
Grind the tissue well in each tube using a sterile pestle.
Heat the tubes at 65 °C for at least 00:15:00 . It is fine to leave it in longer. I often use one hour.
15m
Centrifuge the tubes for 00:03:00 at max rpm.
3m
Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. Label your tubes.
Add 200 µL of Protein Precipitation Solution 350mlPromegaCatalog #A7953 to the tube.
Vortex the tube for 00:00:20 .
Centrifuge the tube for 00:06:00 at max rpm.
6m 20s
Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. Label your tubes
Add 600 µL of 100%IsopropanolIBI Scientific to the tube. This precipitates the DNA.
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.
1m
Add 600 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.
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.
16m
Add 30uL of molecular water to the tube.
Your DNA template is now ready for amplification.