Oct 11, 2022
CTAB DNA extraction protocol for fungi
- 1University of Nebraska Lincoln
Protocol Citation: Erin Carr 2022. CTAB DNA extraction protocol for fungi. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldp9qol5b/v1
Manuscript citation:
Carr, E.C., Barton, Q., Grambo, S., Sullivan, M., Renfro, C.M., Kuo, A., Pangilinan, J., Lipzen, A., Keymanesh, K., Savage, E., Barry, K., Grigoriev, I.V., Riekhof, W.R., Harris, S.D., 2021. Deciphering the potential niche of two novel black yeast fungi from a biological soil crust based on their genomes, phenotypes, and melanin regulation.. doi:10.1101/2021.12.03.471027
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: October 11, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 71183
Funders Acknowledgement:
NASA
Grant ID: 80NSSC17K0737
NSF PRFB
Grant ID: 2209217
Abstract
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
Attachments
Materials
Reagents Needed:
Liquid Nitrogen
CTAB Extraction Buffer + PVP
CTAB Precipitation Buffer (pH=7.2-7.6)
24:1 Chloroform: Isoamyl alcohol
100% Isopropanol (ice cold)
70% Ethanol (ice cold)
100% Ethanol (ice cold)
3M Sodium Acetate (pH=5.2)
5M NaCl
UltraPure water or TE buffer
Proteinase K 20mg/mL
RNase A 10 mg/mL
Instruments/Materials Needed:
Bead beating tubes
0.5 mm Glass beads
Dewar for Liquid Nitrogen
Water bath or heat block set to 70° C
1.5mL Microcentrifuge tubes
2.0mL Microcentrifuge tubes
15mL Centrifuge tubes
Fridge and Freezer (4° C and -20° C)
Pipettes
Refrigerated Microcentrifuge
Fungal samples, 0.03g minimum
Recipes:
CTAB Extraction Buffer + PVP
1% w/v CTAB
1M NaCl
100mM Tris
20mM EDTA
*RIGHT BEFORE USE ADD: 1% w/v Polyvinyl pyrrolidone (PVP)
*RIGHT BEFORE USE ADD: 1% of 20mg/mL Proteinase K
CTAB Precipitation buffer:
1% w/v CTAB
50mM Tris-HCl
10mM EDTA
40mM NaCl
*Adjust pH to 7.2-7.6
Preparing Yeast from liquid culture for DNA extraction
Preparing Yeast from liquid culture for DNA extraction
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.
Preparing Yeast from solid culture for DNA extraction
Preparing Yeast from solid culture for 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
Lysing Filamentous fungi
Lysing Filamentous fungi
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.
DNA extraction Protocol (from cell growth to final DNA product):
DNA extraction Protocol (from cell growth to final DNA product):
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
DNA clean-up
DNA clean-up
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