Oct 06, 2022

Public workspaceIsolation of Neurospora crassa genomic DNA

DOI
dx.doi.org/10.17504/protocols.io.yxmvm2kkbg3p/v1
  • 1Texas A&M University
kdcastillo
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DOI: dx.doi.org/10.17504/protocols.io.yxmvm2kkbg3p/v1
Document Citation: kdcastillo 2022. Isolation of Neurospora crassa genomic DNA . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2kkbg3p/v1
License: This is an open access document 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
Created: October 06, 2022
Last Modified: October 06, 2022
Document Integer ID: 70936
Abstract
Here, we describe the isolation of fungal genomic DNA from Neurospora crassa mycelial mats.
FUNGAL GENOMIC DNA ISOLATION (MINIPREP) FROM N. CRASSA MYCELIA MINI-MATS

Materials:
DNA extraction buffer:
To make 50 ml in a 50 mL conical tube:

5 ml 1M Tris-HCl, pH 8.0, 2 ml 0.5M EDTA, 5 ml 5M NaCl, 5 ml 10% SDS, 33 ml H2O

Prepare prior to collecting mycelia for DNA extraction (# will depend on the # of your samples):
  • Labeled 1.5-ml tubes for collecting mini-mats
  • Labeled 1.5 ml microfuge tubes with 500 μl chloroform.
  • Labeled 1.5 ml microfuge tubes with 600 μl isopropanol.

Part 1: COLLECTING THE MYCELIA

  1. If your mats are coming from a tube, vortex the tube quickly.
  2. Using a sterile wooden stick (RNA-free cabinet), collect the mycelia from each tube and lay on a stack of paper towel (they will look like mycelia lollipops).Usually, 7 samples can fit on the length of a regular paper towel.Press with another layer of paper towel to get excess media off.
  3. Transfer each mycelia mat into the pre-labeled 1.5-ml tubes (set 1).
  4. You can freeze them in -20oC or proceed to the extraction process.

Part 2: DNA EXTRACTION

  1. Measure out the total amount of extraction buffer you need, and add Proteinase K (Freezer #4, tubes labeled PK).Each sample gets 500 μl extraction buffer with 3 μl Proteinase K.Just do the math for making a “master mix” depending on the # of samples you have.
  2. Add ~500-503 μl of the buffer/Proteinase K mix to each sample.Vortex for 5 mins (using the vortex mixer with the round holder for multiple samples or the one which holds racks).Incubate the samples at 65oC for 1 hr (or more, not that critical).
  3. Place tubes on ice, then add 200 μl of 7.5M ammonium acetate. Vortex for 1 min.Place on ice or 4cC fridge for 5 min.
  4. Centrifuge at max speed for 5 min.
  5. Transfer supernatant to TUBE 2 (set with 500 μl chloroform).Vortex for 5 mins. Centrifuge at max speed for 5 min. * Tubes with chloroform go into the phenol/chloroform orange waste bucket next to chemical hood.
  6. While avoiding the interface, transfer ~500 μl of the supernatant into TUBE 3 (set with 600 μl isopropanol).Mix by inversion, then let sit for 10 min. Alternatively, can let sit at room temp. overnight for higher yield.
  7. Centrifuge at max speed for 10 min.
  8. Discard supernatant.Wash pellet with 70% ethanol. Centrifuge at max speed for 5 mins.
  9. Take out the ethanol. Air dry or speed vac.
  10. Suspend in 100 μl 1X TE with 1μl RNAse A (Freezer #4).Once again, you can make a “master mix” of TE buffer and RNase A depending on the number of your samples.Put in the 37oC water bath for 1 hr.
  11. Centrifuge at max speed for 1 min. Quantitate DNA concentration with NanoDrop with 1X TE buffer as blank.