PCR amplification (50Ál volumes) is carried
out on chromosomal DNA using Qiagen Taq polymerase with an
intial denaturation time of 4 minutes at 95oC, followed by
30 cycles of 95oC for 30 seconds, 62oC for 30 seconds and
72oC for 60 seconds. Samples are then maintained at 72oC for
a further 10 minutes, cooled to 4oC and stored at -20oC .
As the same primers are used for amplification and sequencing,
it is important that only a single DNA fragment is amplified
in the initial PCR. This may involve some optimisation of
the annealing temperature. As this organism has a high G+C
content, the use of additives (i.e. DMSO
in conjunction with betaine) may help to improve the
amplification of the PCR fragments from strains where normal
PCR conditions give inadequate amplification.
The DNA fragments are purified using QIAquick (Qiagen) and
sequencing reactions are carried out, in each direction, using
the primers that were used for the initial PCR amplification.
The samples are applied to an automated DNA sequencer with
d-Rhodamine-labeled terminators (PE Applied Biosystems).
Click the name below to obtain a correctly trimmed sequence
for that locus