SUPPLEMENTAL MATERIALS AND METHODS
PREPARATION OF NEUROSPORA GENOMIC DNA
The procedure is based on methods previously described (LUO et al. 1995; OAKLEY et al.
1987). Neurospora mycelia from a 3-day culture grown in 25 ml of 1x Vogel’s minimal
medium/2% sucrose were harvested onto Whatman 541 filter paper discs, lyophilized
overnight, pulverized and resuspended in salt-detergent solution (4% sodium
deoxycholate, 10% Brij 58, 2 M NaCl; 1 ml per 1 g of wet weight). The suspension was
incubated at room temperature for 20 min while mixing end-over-end and then
centrifuged for 10 min at 4°C at 7,670 x g . The clarified supernatant was transferred to
microcentrifuge tube, mixed with 1.2 mL of 2.25 M TCA/50% ethanol and kept at -20°C
for at least 30 min. Genomic DNA was pelleted by centrifugation, washed with 70%
ethanol and resuspended in 200 L of 10 mM NH4OAc with 0.15 mg/mL RNaseA. The
suspension was incubated at 50°C for 1 hour, gently mixed with a vortexer every 15 min,
and then extracted with 200 L of chloroform. The aqueous phase was mixed with 107
L of 7.5 M ammonium acetate and 800 L of isopropanol, the DNA pelleted, washed
with 70% ethanol, briefly dried and resuspended in 100 L of TE. DNA concentration
was determined using a fluorometer.
CONSTRUCTION OF COSMID VECTOR PMLF4
One hundred nanograms of cosmid vector pMLF2 (AN et al. 1996) were digested with
EcoRI and XbaI, filled-in with Klenow polymerase, and ligated in a large volume (100
µl) to promote recircularization. The resulting deletion-derivative was then digested with
BamHI and ligated to an adaptor created by annealing together the oligonucleotide
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primers, pMLF/EcoRV-1 and pMLF/EcoRV-2 (Supplemental Table 1). The ligation mix
was then transformed into E.coli DH5. The resulting vector is 5.5 kb in size and was
named pMLF4.
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