Ultra-fast preparation of gDNA from Fusarium (Gibberella)
for screening of transformants.
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.
approximately 2 mg of mycelium (use sterile pipette tip or toothpick)
of 10xTE buffer.
sample for 50 seconds in a microwave oven at maximum effect.
sample rest for 2 minutes at room temperature (25 oC).
cells for 5 minutes at 10.000 rpm in a table top centrifuge.
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)
For PCR reactions use
of the supernatant.
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.
Tendulkar S.R., Gupta A. and Chattoo B.B “A Simple protocol for
isolation of fungal DNA” in Biotechnology Letters (2003) Vol. 25, p.
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Dette sted blev sidst opdateret
14. July 2010