Large scale DNA preparation

ID: 108

Group:

Fungal Molecular Biology

Prepared by:

Paul Bowyer

Detail:

This protocol was designed to extract high molecular weight DNA for restriction fragment length polymorphism analysis of A.fumigatus isolates (Denning et al., 1990). The protocol is applied to protoplasted mycelia and produces DNA of high purity with fragments of greater than 60 kb.

Year prepared: NULL

Date uploaded: 2010-10-06 22:32:18

Introduction

This protocol was designed to extract high molecular weight DNA for restriction fragment length polymorphism analysis of A.fumigatus isolates (Denning et al., 1990). The protocol is applied to protoplasted mycelia and produces DNA of high purity with fragments of greater than 60 kb.

Materials

  • Modified Vogel’s minimal medium: Vogel’s salts in 1% glucose
  • 0.6 M MgSO4
  • Novozyme buffer:
    • 1.2 M MgSO4
    • 10 mM potassium phosphate pH 5.8
  • Novozyme lysing enzyme (Interspex)
  • Separation buffer A:
    • 0.6 M sorbitol
    • 100 mM Tris-Cl pH 7.0
  • Separation buffer B:
    • 1.2 M sorbitol
    • 10 mM Tris-Cl pH 7.5
  • Lysis buffer:
    • 2% (w/v) SDS
    • 50 mM EDTA
  • Proteinase K stock 100 mg/ml
  • TE buffer:
    • 10 mM Tris-Cl pH 7.5
    • 1 mM EDTA
  • Phenol equilibrated with Tris buffer pH > 7.3
  • Chloroform/isoamyl alcohol (24:1)
  • Dialysis tubing (10 x 6.4 mm molecular weight cutoff 12000-14000).

Equipment

  • Water bath / heating block 70°C
  • Water bath / heating block 50°C
  • Vortex mixer
  • Rotary mixer
  • Water bath (shaking) at 33°C
  • Microcentrifuge and tubes
  • Whatmann 54 paper
  • Buckner funnel
  • Benchtop centrifuge
  • Universal tubes

Procedure

1) Inoculate 50 ml of Vogel’s minimal medium in a 250 ml conical flask with a stock spore suspension 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 the mycelium onto Whatmann 54 paper using a Buckner funnel and a side-arm flask attached to a vacuum pump and wash with 0.6 M MgSO4.  Resuspend in Novozyme buffer to give 40 mg mycelium per ml buffer in a conical flask.

3) Add Novozyme to a final concentration of 2 mg/ml and mix.  Incubate at 33°C  with shaking (100 rpm) for 1-3 h to generate protoplasts.

4) Overlay the protoplast suspension with separation buffer A and centrifuge at 1500 x g at room temperature for 15 min.  The protoplasts will form an interface layer.

5) Remove the interface protoplast layer to a universal tube and make up the volume to 10 ml with separation buffer B.

6) Centrifuge at 1000 x g for 10 min and repeat the 10 ml wash twice.  Resuspend the pellets in 3 ml of separation buffer B.

7) Divide into 3 microcentrifuge tubes, centrifuge at 1000 x g for 10 min and pour off the buffer.

8) Add 1.0 ml of lysis buffer to each microfuge tube.  Dislodge the pellet with the pipette tip and vortex mix for 15 s.

9) Incubate at 70°C for 30 min and centrifuge at 15 000 x g for 5 min.

10) Pool the supernatants from each isolate prep into a 15 ml polypropylene tube, add 0.3 ml of proteinase K to each tube and incubate at 50°C for 1 h.  (The proteinase K digestion may be left O/N at 50°C).

11) Add an equal volume of phenol and place on a rotary mixer for 10 min.  Centrifuge at  1500 x g for 10 min.

12) Remove the aqueous layer to a new tube and add an equal volume of chloroform / isoamyl alcohol.  Invert the tubes several times and centrifuge for 10 min at 1500 x g.

13) Transfer the aqueous layer into pre-washed dialysis tubing and dialyse for 3 days at room temperature against eight changes of TE buffer.  Store samples at 4°C

Procedure Timetable

Fungal culture Step 1 24 hours
Protoplast production Steps 2 – 7 3 – 5 hours
DNA extraction Steps 8 – 12 3 hours
Dialysis Steps 13 3 days

Tips and general comments

1) Protoplast production is affected by several variables and this has been investigated in detail by Birch et al., (1998).

2) With A. nidulans we have observed that protoplast formation is most efficient when prepared from a filamentous culture.  Filamentous growth of A. nidulans is easily produced by growth in thin layer culture: 50 µl of a 108 A. nidulans stock spore solution is inoculated into 10 ml of YEPD broth in a 35 cm petri dish and incubated O/N at 37°C.

3) To shorten the prep. an ethanol precipitation can be performed instead of the dialysis.  The DNA will not be of such high purity and will also suffer from additional shearing.

4) We have attempted DNA extractions using a CTAB method (Moller et al., 1992), but the DNA could not be restricted with endonucleases.  Inhibition of PCR has also been reported when DNA is extracted with this method (Weston et al., 1998).  It is thought that contaminating carbohydrate causes the inhibition of enzyme reactions.

References

Birch M, Nolard N, Shankland GS, Denning DW. DNA typing of epidemiologically-related isolates of Aspergillus fumigatus. Epidemiol Infect 1995;114(1):161-168.

Denning DW, Clemons KV, Hanson LA, Stevens DA. Restriction endonuclease analysis of total cellular DNA of Aspergillus fumigatus isolates of geographically and epidemiologically diverse origin. J Infect Dis 1990;162:1151-1158.

Moller EM, Bahnweg G, Sandermann H, Geiger HH. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 1992;20(22):6115-6116.

Weston G, Nicholson P, Simpson D. Extraction of genomic DNA from maize kernels using the Wizard® genomic DNA purification kit. Promega Profiles 1998;37:6

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

Solutions

1) Novozyme buffer: dissolve MgSO4 in Millipore water and adjust pH to 5.8 using 0.5 M stock solutions of potassium phosphate – use 0.27 ml of KH2PO4 and 1.73 ml of K2HPO4 for every 100 ml.  Filter sterilise (autoclaving causes precipitation)

2) Protoplast separation buffer A: this solution needs to be filter sterilised and stored at 4°C

3) Protoplast separation buffer B: dissolve the sorbitol and make up almost to the correct volume; autoclave (15′), add theappropriate volumes of 1M Tris-Cl, pH 7.5 and 0.5 M EDTA and make up to volume with sterile water.  Store at 4°C.  Sorbitol can not be autoclaved in the presence of TE as the solution will be become discoloured.

Laboratory Protocols