A simple modification of the Aspergillus nidulans transformation protocol increases the transformation frequency
Jane A. Cockram and Heather M. Sealy-Lewis. Department of Biological Sciences, University of Hull, Hull. HU6 7RX
After transformation of Aspergillus nidulans with plasmid DNA the transformants are usually incubated at 37C until transformants appear. We have found that pre-incubation of the transformation plates at room temperature for 24h leads to increased transformation frequencies.
The results of a typical experiment are shown in Table 1. Details of the protocol not specifically mentioned here are the same as described in Tilburn et al. Strain H1326: biA1; argB2; pyroA4; alc500 was inoculated into Aspergillus glucose minimal medium with appropriate supplementation and grown for 16 hours at 25C. The mycelium was divided into two portions one of which was treated with Novozyme 234 for 1.5 h at 30C and the other for 1.8h at 25C. After the protoplast washing procedures the two batches of protoplasts were transformed with pBluearg (constructed by inserting the BamHI/PstI fragment from pILJ16 containing argB into Bluescript). After transformation and plating out of the protoplasts on selective media lacking arginine, half the plates from each treatment were immediately incubated at 37C, and the other half were left at room temperature for 24h before being transferred to the 37C incubator. As can be seen from Table 1 the plates that were pre-incubated at room temperature result in at least a 2-fold increase in the transformation frequency. This result has been repeated consistently in our laboratory. This is a simple modification of the protocol and with some plasmids/strains that are difficult to transform any modifiation that increases the frequency may be beneficial. Lowering the protoplasting temperature also seemed to lead to increased transformation frequencies in this experiment.
Table 1. Transformation experiment testing protoplasting temperature and regeneration conditions on the transformation frequency
|25C||24h at room temp
then at 37C
|25C||37C from start||20|
|30C||24h at room temp
then at 37C
|30C||37C from start||7|
Last modified 8/3/00 KMC