DNA extraction: miniprep

Detail:

This protocol can be performed directly on ground mycelia in a microfuge tube. It provides DNA of sufficient quality for PCR and most restriction digests.

Year prepared: 1956

Introduction

This protocol can be performed directly on ground mycelia in a microfuge tube. It provides DNA of sufficient quality for PCR and most restriction digests.

Materials

  • Vogel’s minimal medium (Vogel, 1956)
  • Extraction buffer: 0.7 M NaCl
    • 0.1 M Na2(SO3)
    • 0.1 M Tris-Cl pH 7.5
    • 0.05 M EDTA
    • 1%(w/v) SDS
  • Chloroform /isoamyl alcohol (24:1)
  • Isopropanol
  • 7.5 M ammonium acetate
  • 70% ethanol
  • TE buffer:
    • 10 mM Tris-Cl pH 7.5
    • 1 mM EDTA
  • Liquid nitrogen

    Equipment

  • Pestle and mortar
  • Microcentrifuge tubes
  • Heating block / water bath at 65° C
  • Microcentrifuge
  • Vortex mixer

Procedure

  1. Inoculate 50 ml of Vogel’s minimal medium to a final concentration of 107 spores / ml and incubate with shaking at 200 rpm until late exponential phase (18-24 h) at 37° C.
  2. Dry down the mycelium onto Whatmann 54 paper using a Buckner funnel and a side-arm flask attached to a vacuum pump (need ~0.5 g wet weight) and wash with 0.6 M MgSO4. Continue with the prep or freeze-dry the mycelium for extraction at a later date.
  3. Grind freeze-dried mycelium in a mortar OR with fresh mycelium add liquid nitrogen and grind in a -20° C cooled mortar. Add the powder to a 1.5 ml microcentrifuge tube using an ethanol-cleaned spatula – add no more than 0.4 ml (use 2 or 3 tubes if necessary).
  4. Add 0.6 ml of extraction buffer (heated to 65° C) to the microfuge tube and incubate at 65° C for 20 min.
  5. Add 0.6 ml of chloroform / isoamyl alcohol to the tube, vortex mix thoroughly and incubate on ice for 30 min.
  6. Centrifuge at 12,000 x g for 30 min and carefully transfer the aqueous phase to a fresh microfuge tube without disturbing the interface. Add an equal volume of isopropanol, mix by inversion and incubate at room temperature for 10 minutes. A stringy clump of DNA should become visible.
  7. Centrifuge at 2000 x g for 5 min, remove supernatant and allow the pellet to air dry. Resuspend the pellet in 200 µl of 18 megaohm water. Incubation at 37° C for 15-30 min with occasional flicking aids resuspension. Alternatively, the DNA can be left to resuspend O/N at 4° C.
  8. Add 100 µl of ammonium acetate, mix by inversion and incubate on ice for 1 hour.
  9. Centrifuge at 12000 x g for 30 min, transfer the supernatant to a fresh tube and add a 0.54 volumes of isopropanol, mix by inversion and incubate at room temperature for 10 minutes. DNA should visibly precipitate out of solution and should be cleaner than in step 6.
  10. Centrifuge at high speed for 5 min, remove supernatant and wash the pellet in 500 µl of 70 % ethanol. Centrifuge at high speed for 5 min and remove all the ethanol. Allow the pellet to air dry, but do not dry out completely.
  11. Resuspend the DNA pellet in 100 µl of TE or 18 megaohm water. Wash the tube around the position of the pellet to improve recovery of DNA. Incubate at 37° C or leave at 4° C O/N to aid resuspension.

Timetable

Fungal culture (1) 24 h

Freeze drying (2) 12 h

DNA extraction (3-11) 5-6 h with 2 possible O/N steps

Tips and general comments

  1. This prep. can yield substantial quantities of RNA and the DNA needs to be treated with (1 µl of 1mg / ml stock) RNase A before use.
  2. It is generally better to put a little bit less rather than a little bit more ground-up mycelium in a microcentrifuge tube. Final yields will generally be improved if the prep. is spread out over several tubes.
  3. All of the incubation steps on ice can be left for longer if desired and the 10 min DNA precipitations at RT (6 and 9) can also be left for longer on ice if this is more convenient.

References

Vogel HJ. (1956) A convenient growth medium for Neurospora (medium N). Microbiol. Gen. Bull. 13:42-44

Laboratory Protocols