DNA Isolation

2.5.1 Plasmid DNA extraction

To extract plasmid DNA for routine screening, the boiling miniprep method, adapted from (Holmes and Quigley, 1981), was used. Cultures were grown at 37°C

with shaking at 200 rpm for approximately 18 hours in LB broth, then 1.5 mL harvested by centrifugation. Pellets were resuspended in 350 μL of HQ-STET buffer (8% [w/v] sucrose, 50 mM EDTA [pH 8], 5 mM Tris [pH8], 5% [w/v] TritonX-100). 25 μL of lysozyme (10 mg/mL in HQ-STET buffer) was added to each tube and the tubes were boiled for 40 s. Immediately after the boiling step, the tubes were centrifuged at 15000 × g for 10 min and the resulting gelatinous pellet was removed and discarded. Plasmid DNA was then precipitated by the addition of 450 μL of isopropanol followed by centrifugation at 15000 × g for 5 min. DNA pellets were washed in 70% ethanol, air dried and resuspended in 50 μL of filter-sterile Milli-Q water.

High quality plasmid DNA preparations to be used for sub-cloning, transformation or as templates for automated sequencing were prepared using either the QIAprep Spin Miniprep or Midiprep kits (Qiagen), according to the instruction manuals supplied by the manufacturers.

2.5.2 Genomic DNA Isolation

Fungal genomic DNA was extracted using methods adapted from Byrd et al., (1990), Al-Samarrai and Schmid, (2000) and Wu et al., (1995).

For routine screening of fungal transformants, a method adapted from (Wu et al., 1995) was used to extract small amounts of genomic DNA. Fungal mycelium was cultured in PD broth for three days in 1.5 mL tubes, pelleted by centrifugation and freeze-dried, then ground to a fine powder using a micropestle. 150 μL of lysis buffer (100 mM Tris [pH 8], 100 mM EDTA [pH 8], 1% [w/v] SDS) was added, mixed by pipetting and incubated at 70°C for 30 min. Lysates were mixed with 150 μL of 5 M potassium acetate, incubated on ice for 10 min and centrifuged at 15000 × g for 10 min. DNA was precipitated from the supernatant by addition of 0.6 volumes isopropanol and centrifugation at 15000 × g for 15 min. DNA pellets were washed twice with 70% ethanol, air-dried and resuspended in 20 μL filter-sterile Milli-Q water.

Large amounts of genomic DNA for Southern analysis were prepared using the method of either Byrd (1990) or Al-Samarrai (2000). For the Byrd method, 30 mg of freeze-dried mycelium was ground to a fine powder under liquid N2 and

suspended thoroughly in 500 μL of extraction buffer (150 mM EDTA [pH 8], 50 mM Tris [pH 8], 1% sodium lauroyl sarcosine, 2 mg/mL proteinase K). The solution was incubated at 37°C for 20 min then at 65°C for 1 – 2 h followed by centrifugation at 15000 × g for 10 min. The supernatant was removed, mixed with an equal volume of phenol/chloroform (1:1 v/v) and centrifuged at 15000 × g for 10 min. The aqueous phase was removed and the procedure repeated until the aqueous phase was clear, then the aqueous phase was mixed with an equal volume of chloroform and centrifuged at 15000 × g for 10 min. To the aqueous phase obtained from the chloroform extraction, an equal volume of isopropanol was added, mixed by inversion and incubated at –20°C for 20 min followed by centrifugation at 15000 × g for 10 min. To remove remaining polysaccharides, the pellet was resuspended in 1 M NaCl and centrifuged at 15000 × g for 10 min, DNA was then precipitated from the supernatant by mixing with an equal volume of isopropanol, incubation at – 20°C for 10 min and centrifugation at 15000 × g for 10 min. DNA pellets were washed three times with 70% ethanol, air-dried and resuspended in 50 μL TE buffer (10 mM Tris [pH 8], 0.1 mM EDTA [pH8]).

For the Al-Samarrai method, 30 mg freeze-dried mycelium was ground to a fine powder under liquid N2 and resuspended in 500 μL lysis buffer (40 mM Tris [pH 8],

20 mM sodium acetate, 1 mM EDTA [pH 8], 1% [w/v] SDS) by approximately 30 cycles of vigorous pipetting, until the viscosity was significantly reduced. 2 μL RNase A (10 mg/mL) was added and the mixture incubated for 5 min at 37°C. Cell debris was removed by addition of 165 μL of 5 M NaCl, inversion and centrifugation at 15000 × g for 20 min. The supernatant was removed, mixed with an equal volume of phenol/chloroform (1:1 v/v) and centrifuged at 15000 × g for 20 min. The aqueous phase was removed and the procedure repeated with chloroform until the aqueous phase was clear. DNA was precipitated from the aqueous phase by addition of 0.1 volumes of 3 M sodium acetate and 2.5 volumes of 96% ethanol.

DNA pellets were washed three times with 70% ethanol, air-dried and resuspended in 50 μL TE buffer (10 mM Tris [pH 8], 0.1 mM EDTA [pH8]).

2.5.5 Chromosomal DNA Isolation in Agarose Plugs

To prepare chromosomal DNA for pulsed-field gel electrophoresis, fungal protoplasts (section 2.13.1) were resuspended in STC buffer (1 M sorbitol, 50 mM CaCl2, 50 mM Tris [pH 8.0]) to a concentration of 1 × 109/mL, mixed with an equal

volume of 1.4% low melting point agarose in GMB buffer (0.9 M sorbitol, 125 mM EDTA [pH 7.5]) and allowed to set in plug moulds. The plugs were then incubated at 50°C in SE buffer (2% [w/v] SDS, 250 mM EDTA [pH 8]) for 18 h, followed by 24 h at 50°C in 10 mL 10 x ET buffer (10 mM Tris [pH 8], 500 mM EDTA [pH 8]) with 20 mg of proteinase K (Invitrogen) and 100 mg of sodium lauroylsarcosine (Sigma). Plugs were finally washed four times with 1 x ET buffer and stored in 1 x ET buffer at 4°C.

2.5.4 λ DNA Isolation

Bacteriophage λ DNA was isolated from plate lysates using PEG precipitation and phenol/chloroform purification based on the method in Sambrook et al., (1989). Recombinant phage were plated for confluent lysis on LB agarose (1.5%) plates, overlaid with 5 mL of SM buffer (100 mM NaCl, 8 mM MgSO4.7H2O, 0.01% (w/v)

gelatine, 50 mM Tris [pH 8]) and left overnight at 4°C. The lysate was collected and treated with DNase and RNase (1 μg/mL final) and incubated at 37°C for 30 min. 5 mL of PEG solution (20% PEG 6000 (w/v), 2 M NaCl) was added, the solution mixed and incubated on ice for 1 h. Phage were pelleted by centrifugation at 5800 g for 30 min at 4°C, resuspended in 500 μL of SM buffer with 5 μL of 10% (w/v) SDS and 10 μL of 250 mM EDTA (pH 8), and incubated at 68°C for 15 min. To remove phage proteins, an equal volume of phenol was added, the mixture was vortexed for 10 s, incubated at room temperature for 5 min and centrifuged at 15000 × g for 5 min. The aqueous phase was removed and the procedure repeated with phenol/chloroform (1:1 [v/v]), then chloroform. To the aqueous phase obtained from the chloroform extraction, an equal volume of isopropanol was added, mixed

× g for 10 min. DNA pellets were washed with 70% ethanol, air dried and resuspended in 50 μL TE buffer (10 mM Tris [pH 8], 0.1 mM EDTA [pH8]).