TITLE: CTAB DNA extraction protocol for fungi
AUTHORS: Erin Carr

[DESCRIPTION]
CTAB Fungal DNA Extraction Protocol
Erin Carr- University of Nebraska-Lincoln
*Adapted from Cubero et al., 1999 and Lagonigro et al., 2004
                                                                    February 2020; Updated August 15th, 2022

[STEPS]
SECTION: Preparing Yeast from liquid culture for DNA extraction
1. Grow fungi in preferred media type till fungi are in mid-exponential phase
SECTION: Preparing Yeast from liquid culture for DNA extraction
2. Centrifuge fungal samples at 10,000x g for 1 minute and wash with water 3 times, centrifuging in between, then obtain as tight of a fungal pellet as possible and remove all liquid from the tube.
SECTION: Preparing Yeast from solid culture for DNA extraction
4. Grow fungi on preferred media type till fungi grows to its maximum on the plate
SECTION: Preparing Yeast from solid culture for DNA extraction
5. Place 800µL of water in a 1.5mL tube and weigh recording the weight
SECTION: Preparing Yeast from solid culture for DNA extraction
6. Scrape 0.03g of fungus off the plate and place into the tube, avoiding agar
SECTION: Preparing Yeast from solid culture for DNA extraction
7. Centrifuge at 10,000x for 1 minute to obtain a fungal pellet and remove all water 
SECTION: Lysing Filamentous fungi
9. Grab as many mortar and pestles as necessary for the number of samples you have (or wash one out thoroughly with 70% ethanol between samples)
SECTION: Lysing Filamentous fungi
10. Obtain liquid nitrogen placed in Dewar
SECTION: Lysing Filamentous fungi
11. Place tubes with processed fungi in a small container and pour liquid nitrogen over the tubes until the fungi pellets are frozen completely
SECTION: Lysing Filamentous fungi
12. With tube lids closed, tap the lids against the table to dislodge the pellet from the tube
SECTION: Lysing Filamentous fungi
13. Put the frozen fungal pellet into the mortar and pour additional sterile Liquid nitrogen over the pellet
SECTION: Lysing Filamentous fungi
14. Carefully grind the fungus being sure not to launch it out of the mortar, grind till the fungus is a powder, adding more liquid nitrogen if the fungus gets too liquidy
SECTION: Lysing Filamentous fungi
15. Scrape ground up fungus out of the mortar and place in a fresh 1.5mL tube, place sample in -80° C freezer until ready for DNA extraction.
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
16. Grow fungi in preferred media type till fungi are in mid-exponential phase (3-7 days)
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
17. Pipette 1 mL of fungus into a 1.5 mL tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
18. Centrifuge fungal samples at 10,000x g for 1 minute
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
19. Wash with 1 mL of ddH2O 3 times, centrifuging in between, then obtain as tight of a fungal pellet as possible and remove all liquid from the tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
20. Take your 1.5 mL tube of washed cell pellet and place in liquid nitrogen to flash freeze and place tubes in -80 freezer till you’re ready to extract DNA
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
22. Turn on water bath/heat block to 70° C
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
23. Determine the number of samples you will be processing, multiply that number by 750 µL to determine the amount of CTAB Extraction buffer you will need (700 µL per sample) and aliquot that out into a 15 mL centrifuge tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
24. Add 1% w/v PVP (.01g/mL) and 1% v/v 20mg/mL Proteinase K (5µL/500µL) to your aliquoted CTAB Extraction buffer
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
25. Grab as many bead beating tubes as you have samples
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
26. Pour ~300uL of glass beads into tubes and label tubes accordingly
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
27. Remove tubes of frozen fungus from the freezer
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
28. Dislodge pellet from bottom of tube by pipetting 700 µL of the complete CTAB Extraction buffer into each frozen sample tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
29. Transfer sample in CTAB Extraction buffer to Bead beating tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
30. Put tubes in 70 °C water bath or heat block for 5 minutes, then bead beat for 5 minutes; repeat 2 more times (30 mins total) (change temperature of heat block/water bath to 37° C after this step, unless you have a 37° C incubator)
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
31. Confirm cells have lysed via microscopy, 5 µL of sample onto a microscope slide. If cells have not lysed, bead beat for additional 2-5 minutes.
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
32. Remove tubes from the water bath/heat block and transfer as much liquid as possible into a new 2 mL microcentrifuge tube, avoiding the glass beads
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
33. Add 1x the volume of 24:1 Chloroform: Isoamyl to the tubes, invert tubes to mix and then centrifuge tubes at 10,000x g for 5 minutes
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
34. Carefully remove tubes from centrifuge, collect upper layer only, and transfer the upper layer into a new 2 mL microcentrifuge tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
35. Add 2 volumes of CTAB Precipitation buffer to the tubes and mix by inverting for 2 minutes
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
36. Centrifuge tubes at 13,000x g for 15 minutes
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
37. Remove supernatant, being sure to leave the pellet behind in the tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
38. Re-suspend pellet in 350 µL of 5M NaCl
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
39. Add 2µL of 10 mg/mL RNase A to tubes and place tubes at 37° C for 30 minutes
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
40. Remove tubes from 37° C and add one volume of 24:1 Chloroform: Isoamyl; mix thoroughly by inverting
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
41. Centrifuge tubes at 10,000x g for 5 minutes
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
42. Remove upper phase and transfer to a new 1.5 mL tube
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
43. 31.  Add 6 volumes of ice cold isopropanol and mix by inverting
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
44. (PAUSE POINT) Place tubes in -20 °C freezer for 15 minutes OR OVERNIGHT/FOR MULTIPLE DAYS
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
45. Centrifuge tubes in 4 °C at 13,000x g for 20 mins
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
46. Remove all liquid and let air dry for 30 mins to over night, until it’s dry to remove excess alcohol
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
47. Resuspend pellet in 30-50 µL of UltraPure water or TE buffer 
SECTION: DNA clean-up
48. Add 0.1 volumes of 3M Sodium Acetate and 3 volumes of ice cold 100% Ethanol to your DNA samples, mix by inverting.
SECTION: DNA clean-up
49. Freeze samples at -80° C for 20mins or at -20° C overnight.
SECTION: DNA clean-up
50. Centrifuge tubes at 4° C at max speed for 30 minutes
SECTION: DNA clean-up
51. Remove supernatant and resuspend pellet in 1mL of ice cold 70% Ethanol, being sure the DNA pellet becomes loose from the tube and floats freely in the liquid.
SECTION: DNA clean-up
52. Centrifuge at 4° C  at max speed for 10 minutes
SECTION: DNA clean-up
53. Remove Ethanol and let tubes dry completely with no ethanol left in the tubes. Overnight with running air over the open tubes works best.
SECTION: DNA clean-up
54. Pre-heat elution buffer (TE or water) at 65° C, then re-suspend DNA in 30-50µL of warmed elution buffer
SECTION: Preparing Yeast from liquid culture for DNA extraction
3. Place tube of cell pellet in liquid nitrogen to freeze cells, then keep tube in -80 freezer until you are ready to perform the DNA extraction.
SECTION: Preparing Yeast from solid culture for DNA extraction
8. Place tube in liquid nitrogen to flash freeze, and place frozen tube in -80 freezer until you are ready to do the DNA extraction
SECTION: DNA extraction Protocol (from cell growth to final DNA product):
21. START OF DNA EXTRACTION