CTAB DNA extraction protocol for fungi

Grow fungi in preferred media type till fungi are in mid-exponential phase

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.

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.

Grow fungi on preferred media type till fungi grows to its maximum on the plate

Place 800µL of water in a 1.5mL tube and weigh recording the weight

Scrape 0.03g of fungus off the plate and place into the tube, avoiding agar

Centrifuge at 10,000x for 1 minute to obtain a fungal pellet and remove all water

Place tube in liquid nitrogen to flash freeze, and place frozen tube in -80 freezer until you are ready to do the DNA extraction

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)

Obtain liquid nitrogen placed in Dewar

Place tubes with processed fungi in a small container and pour liquid nitrogen over the tubes until the fungi pellets are frozen completely

With tube lids closed, tap the lids against the table to dislodge the pellet from the tube

Put the frozen fungal pellet into the mortar and pour additional sterile Liquid nitrogen over the pellet

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

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.

Grow fungi in preferred media type till fungi are in mid-exponential phase (3-7 days)

Pipette 1 mL of fungus into a 1.5 mL tube

Centrifuge fungal samples at 10,000x g for 1 minute

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

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

START OF DNA EXTRACTION

Turn on water bath/heat block to 70° C

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

Add 1% w/v PVP (.01g/mL) and 1% v/v 20mg/mL Proteinase K (5µL/500µL) to your aliquoted CTAB Extraction buffer

Grab as many bead beating tubes as you have samples

Pour ~300uL of glass beads into tubes and label tubes accordingly

Remove tubes of frozen fungus from the freezer

Dislodge pellet from bottom of tube by pipetting 700 µL of the complete CTAB Extraction buffer into each frozen sample tube

Transfer sample in CTAB Extraction buffer to Bead beating tube

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)

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.

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

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

Carefully remove tubes from centrifuge, collect upper layer only, and transfer the upper layer into a new 2 mL microcentrifuge tube

Add 2 volumes of CTAB Precipitation buffer to the tubes and mix by inverting for 2 minutes

Centrifuge tubes at 13,000x g for 15 minutes

Remove supernatant, being sure to leave the pellet behind in the tube

Re-suspend pellet in 350 µL of 5M NaCl

Add 2µL of 10 mg/mL RNase A to tubes and place tubes at 37° C for 30 minutes

Remove tubes from 37° C and add one volume of 24:1 Chloroform: Isoamyl; mix thoroughly by inverting

Centrifuge tubes at 10,000x g for 5 minutes

Remove upper phase and transfer to a new 1.5 mL tube

31. Add 6 volumes of ice cold isopropanol and mix by inverting

(PAUSE POINT) Place tubes in -20 °C freezer for 15 minutes OR OVERNIGHT/FOR MULTIPLE DAYS

Centrifuge tubes in 4 °C at 13,000x g for 20 mins

Remove all liquid and let air dry for 30 mins to over night, until it’s dry to remove excess alcohol

Resuspend pellet in 30-50 µL of UltraPure water or TE buffer

Add 0.1 volumes of 3M Sodium Acetate and 3 volumes of ice cold 100% Ethanol to your DNA samples, mix by inverting.

Freeze samples at -80° C for 20mins or at -20° C overnight.

Centrifuge tubes at 4° C at max speed for 30 minutes

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.

Centrifuge at 4° C  at max speed for 10 minutes

Remove Ethanol and let tubes dry completely with no ethanol left in the tubes. Overnight with running air over the open tubes works best.

Pre-heat elution buffer (TE or water) at 65° C, then re-suspend DNA in 30-50µL of warmed elution buffer