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DATA ANALYSIS
DATABASES
B.burgdorferi
B.cereus
B.henselae
B.pseudomallei
C.albicans
C.glabrata
C.krusei
C.tropicalis
C.jejuni
C.neoformans var grubii
E.coli
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Leptospira spp.
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S.pyogenes
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V.vulnificus
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PCR conditions

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

ace | gltB | gmhD | lepA | lipA | narK | ndh

 
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