Fungal strain and culture conditions
The Trichoderma reesei strain QM9414 (ATCC number 26921) was transformed for Hygromycin B resistance, as described in [11]. Five-day-old spores of two transformants (TR1 and TR2) were harvested from Potato Dextrose Agar (PDA) slants with 3 ml of sterile water, resulting in a dense spore suspension (109-1010 spores/ml). 40 μl of this suspension was placed on a small 2,2 cm cellophane disk (BIO-RAD#165-0963) sterilized by autoclaving in water and placed on the surface of a solid medium (Potato Dextrose Agar). The culture was then left overnight to grow for 14 hours at 28°C. When a large number of samples was analyzed we grown the fungus on 12 wells cell culture plates.
DNA Purification
Following the fungal growth, the cellophane disk was placed in a 1,5 ml microcentrifuge tube containing 500 μl of a ressuspension buffer (50 mM Tris-HCl, pH 7.5; 10 mM EDTA; 1% SDS). After vigorous whirling, the mycelia pellicle grown on the cellophane disk was readily detached and the cellophane disk removed from the tube. A small volume (200 μl, measured in the tube) of glass beads (Sigma) was added to the tube, which was then vigorously mixed in a vortex for 10 seconds and left to rest for 15 seconds on ice. This step was repeated twice, after which 400 μl of phenol-chloroform was added. The tube was placed in a shaker in a horizontal position and shaken at 200 RPM (37°C) for 15 minutes. Immediately after this procedure, the tube was centrifuged at 15.000 g for 5 minutes, the top aqueous phase (~400 μl) transferred to a new tube and the DNA precipitated by the addition of a 1/10 volume (~40 μl) of 3 M Sodium Acetate, pH 5.8 and 1 ml of cold absolute ethanol. The tube was placed in a freezer at -20°C for 30 minutes and then centrifuged at 15.000 g for 15 minutes, after which the pellet was washed once with 1 ml of ethanol 70% and then with absolute ethanol. After being dried, the DNA pellet was ressuspended in 20 μl of TE-RNAse (20 μg/ml) and quantified spectrophotometrically or by comparison with standards (High Mass DNA ladder – Invitrogen) in a 1% agarose gel. Alternatively and safety, the phenol-chloroform extraction can be suppressed by a direct precipitation after the glass beads step. To the tube still containing the glass beads was added 150 μl of potassium acetate, pH 4.8 (made of 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of milli-Q water). The tube was vortexed briefly and spun at 15.000 g for 2 min. After transferring the supernatant to a new 1.5 ml tube, 600 μl of isopropanol was added. After mixing by inversion the tube was centrifuged at 15.000 g for 10 min. The resultant DNA pellet was washed with 500 μl of 70% ethanol, air dried and ressuspended in 20 μl of TE-RNAse (20 μg/ml).
PCR
The Polymerase Chain Reaction was accomplished according to a modified Saiki et al. protocol [12], using the purified DNAs as templates. The PCR was carried out using internal primers to amplify a 750 pb fragment of the hph gene (resistance to Hygromicin B)[13] present in the Trichoderma reesei transformants used in this work. PCRs were made in 200 μl tubes containing 100 ng of DNA, 10 pmoles of each primer (Forward CCTGAACTCACCGCGACGTCT and Reverse CTCCGGATGCCTCCGCTCGAAGT), 1× PCR buffer (Invitrogen), 1.5 mM MgCl2, 1 U of Taq DNA polymerase (Invitrogen) and Milli-Q water to 25 μl. The reaction was carried out in an MJ Research thermocycler programmed for 30 cycles of 94°C 1 min, 55°C 1 min and 72°C 1.5 min, with 4-min initial and final steps.
Southern blotting
Southern blotting was performed according to Sambrook et al.[14]. Two μg of DNA were subjected to total digestion using 10 U of the restriction enzymes Bam HI and Eco RI for 3 hours. DNA from a non-transformed Trichoderma reesei was included as a control. The digested samples were separated in an 1% agarose gel (TBE buffer) together with a 1 Kb ladder (Invitrogen). The gel's DNA was then transferred to a nylon membrane (Hybon N+ – Amersham Biosciences) which was first hybridized to an α-32PdATP labeled 1 Kb DNA ladder probe (Invitrogen), washed according to the manufacturer's recommendations and rehybridized with an α-32PdATP labeled PCR fragment of the hph gene. Both probes were labeled using the "Ready-to-Go DNA labelling Kit" (Amersham Pharmacia Biotech). After hybridization and washing, the membrane was exposed to X-Ray film (Kodak X-Omat) for 6 hours. The hph probe detected a fragment of approximately 0.8 Kbp.