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Efficient Transfection of Neurospora Crassa

2 ml tubes on ice. In Tube #1, 1 µg of supercoiled DNA (plasmid pCSN44) was added. The selection marker on this plasmid was for hygromycin B resistance [4]. In Tube #2, an equal volume of TE (10 mM TrisCl, 1 mM EDTA, pH 8) was added for control purposes. Both tubes were left on ice, while chilling two 2 mm Eppendorf electroporation cuvettes. The Eppendorf 2510 was set to 2,000 V and the solution from Tube #2 was transferred to a dry, chilled cuvette and then placed in the 2510 (electroporation takes place by pressing the pulse button twice). The cuvette was quickly removed after electroporation and placed on ice. 960 µl of cold sorbitol was added to the cuvette and mixed gently using a Pasteur pipette, then removed and placed in an Eppendorf 2 ml tube. Solution from Tube #1 was then added to a dry, chilled 2 mm cuvette and placed in the 2510 for electroporation. The subsequent steps performed with the sample were identical with the control.

Selection
100 µl of each sample were plated on selective FIGS plates using 10 ml of regeneration agar [5]. The plates were incubated at 30°C for 2 days. Note that these plates must be incubated for at least two days. The number of transfectants was then determined.

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