Ultra-fast preparation of gDNA from Fusarium (Gibberella) for screening of transformants.

I have found that the protocol by Tendulkar et al. also function with Fusarium species. Utilization of this strategy can seriously speed up the screening process as transformation colonies can be tested directly from the originate plates, without the need for prior propagation or large scale purification of genomic DNA. In addition the PCR based screening is much faster than e.g. Southern blotting.  

1. Transfer approximately 2 mg of mycelium (use sterile pipette tip or toothpick) to 50 µl of 10xTE buffer.
2. Cook the sample for 50 seconds in a microwave oven at maximum effect.
3. Let the sample rest for 2 minutes at room temperature (25 ^oC).
4. Spin the cells for 5 minutes at 10.000 rpm in a table top centrifuge.
5. Transfer the supernatant to a new Eppendorf tube and store at -20 ^oC until use. (some times it can be an advantage to store the samples in PCR tubes in strip format as it allows use of multi channel pipettes during the subsequent PCR steps) – However, be careful with the lids as it is easy to cross contaminate the samples by mixing up the lids)
6. For PCR reactions use 1 µl of the supernatant.

The PCR reaction:  The described protocol has been tested with numerous primer sets and the general experience has been that the annealing temperature should be lowered with about 5 ^oC and the number of cycles should be increased with 5-7 cycles.

Remarks:        The method has been tested on F. culmorum, F. graminearum and F. pseudograminearum transformants.