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Efficient Transfection of Neurospora Crassa

be transfected efficiently without having to weaken the cell wall or create protoplasts.

OBJECTIVE:

To provide a simple, efficient method for transfecting N. crassa conidia with the Eppendorf Electroporator 2510.

EXPERIMENTAL METHOD:

Cell Cultivation and Harvesting
A Neurospora crassa wild type strain was cultured in VM agar with appropriate supplements in a 250 ml Erlenmeyer flask (a 125 ml flask may also be used) [3]. The flask was incubated at 30°C for 3 days, then transferred to room temperature and exposed to fluorescent light for an additional 5 days. It is necessary to use conidia that are at least eight days old.

The conidia were harvested using sterile water. The suspension was transferred to a 50 ml conical tube and centrifuged at 2,500 rpm for 5 minutes to pellet the conidia. After aspirating the supernatant, the pellet was re-suspended in 25 ml of sterile water. The tube was vortexed to bring the conidia pellet into solution and then re-centrifuged at 2,500 rpm for 5 minutes. The conidia pellet was re-suspended in 25 ml of cold 1 M sorbitol (vortexed for re-suspension) and then centrifuged as stated above; this was repeated twice.

The final pellet was re-suspended in 0.5 ml cold sorbitol and transferred to a 2 ml Eppendorf microcentrifuge tube. The concentration of the suspension was determined using a hemacytometer. The optimal concentration is approximately 2.5 x 109/ml. It may be necessary to concentrate or dilute the sample to bring it to optimal concentration.

Electroporation Procedure
The suspension was transferred in 40 µl aliquots to two Eppendorf
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