Biology Reference
In-Depth Information
A. DNA Isolation
lysate plate
boiling plate
37°C
inoculate into 96-well
plate (in sterile water)
100°C for 15 mins
in thermocycler
pellet cells at
4000 rpm
dilute cell lysates
(1:40) in new plate
streak 12 strains
on 8 agar plates
scrape cells
with pipet tip
B. PCR Amplification
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C.
spa
Typing
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Fig. 3. High-throughput
spa
-typing protocol.
(A)
Isolation of DNA from boiled
lysates of
Staphylococcus aurus
culture in 96-well format;
(B)
PCR amplification and
verification by agarose gel electrophoresis; and
(C)
DNA sequencing and
spa
-type
assignment.
The cycling procedure lasts for approximately 2 h, following which 5 μL of
the reaction products is run on a 1% agarose gel, stained in ethidium bromide,
and visualized by UV light. The presence of a single amplicon ranging in size
from
100 to 500 bp is evidence of a successful amplification reaction; faintly
staining bands are sufficient to produce quality sequence data. Reactions that
produce multiple bands are suggestive of sample contamination with more than
one strain of
S. aureus
. Additionally, experience in our laboratory has suggested
potential specificity issues with primer TIGR-F; an alternative forward primer
may be useful for samples that fail to amplify as expected. Reactions that fail to
produce amplicons may be reboiled in individual tubes utilizing the water bath
method, diluted further (up to 1:100) in sterile water and reamplified. Samples
that fail to amplify multiple times should be set aside for further analysis and
should be confirmed to be
S. aureus
by other methods, such as isolation on
mannitol salt agar followed by coagulase testing, 16S rRNA sequencing, or
real-time PCR detection of the
spa
gene.
∼
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