Sequence Analysis of the Variable Number Tandem Repeat in Staphylococcus aureus Protein A Gene - Bacterial Pathogenesis: Methods and Protocols - page 274

bridges found in the staphylococcal cell wall. For spa typing, however, a
simple boiling procedure in sterile water is sufficient to isolate enough target
DNA to produce robust amplicons for direct sequencing; for large numbers of
samples, however, greater lysis efficiency may be obtained by prior treatment
with lyso-staphin, as described below.
1. Grow cells overnight on solid media. S. aureus grows well on tryptic soy agar,
Columbia agar, GL agar, 5% sheep blood agar, and mannitol salt agar (a selective
medium on which S. aureus colonies produce yellow halos).
2. Isolate a small amount of bacterial colony using a sterile pipet tip; resuspend the
colony in 100 μL of sterile water in a 1.5-mL tube.
3. Boil contents of tube for 15 min in a water bath and microcentrifuge for 5 min
at maximum speed. The supernatant may be used directly for PCR, or it may be
diluted anywhere from 10- to 100-fold in sterile water. This minimizes the effects
of potential inhibitors or excess template concentration.
3.1.1. High-Throughput DNA Isolation
For higher throughput, up to 12 strains may be streaked in thin lines on
individual agar plates ( see Fig. 3A ), with each plate corresponding to one row
of a 96-well microtiter plate.
1. Aliquot 100 μL of a 100 μg/mL lysostaphin-water mixture into each well; inoculate
with 96 isolates using pipet tips. Mix well.
2. Seal plate and incubate at 37 ° C for 15-30 mins. Boil for 15 mins in water bath,
or place in thermal cycler for 15 mins at 100 ° C.
3. Centrifuge plate at 2500 rpm for 5-10 mins; dilute 5 μL of supernatant (1:40) in
sterile water in a new plate. Use a diluted template for PCR.
3.2. PCR
PCR reactions are typically set up in 30-μL volumes to ensure suffi-
cient volume for gel analysis, amplicon purification, and DNA sequencing
( see Fig. 3B ). Any standard commercial DNA polymerase mixture containing
dNTPs may be utilized. Reaction mixtures include 0.1 μ M of each of
the following primers shown in Fig. 1B : (TIGR-F) 5´-GCCAAAGCGCTA
ACCTTTTA-3´ and (TIGR-R) 5´-TCCAGCTAATAACGCTGCAC-3´.
1. Dispense reaction mixtures in 25 μL volumes into 0.2-mL PCR reaction tubes (or
into a 96-well microtiter plate) to which 5 μL of the 1:40 diluted lysate is then added.
2. Set cycling parameters as follows: An initial 2-min denaturation step at 94 °C,
followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for
1 min, and extension at 72 °C for 1 min. Although the overall amplicon size
ranges from 100-500 bp, the longer annealing time seems to improve product
yield.