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- In S. aureus, standard antibiotic concentrations are 5 μg/mL
tetracycline (Tet marker), 50 μg/mL kanamycin (Kan marker),
or 5 μg/mL erythromycin plus 25 μg/mL lincomycin (Ery
marker). Note that several steps in the transformation and
transduction protocols described herein use altered
concentrations. - “Junction PCR” with one primer outside of your homology
region and a second primer within the plasmid backbone can
be used in colony PCR to analyze these clones. - The lysate from a previous preparation should ideally contain
no selectable markers (i.e., use a lysate grown on the wild type
recipient). If a “wild type” donor phage cannot be obtained,
instead use a phage grown on a strain with a different resis-
tance marker to the one being selected for in this transduction.
These precautions help to prevent the accidental transduction
of incorrect mutations. - If solution does not appear turbid, add more bacterial culture
as required. It is critical that the culture appears slightly cloudy
to be able to judge the next step. - This normally occurs overnight. If the mixture clarifies within
a few hours, add more donor culture until the mixture becomes
turbid, then leave overnight. - It is important to completely drain all liquid from the pellet at
this stage. Ensure this by inverting the tube onto clean tissue
paper and tapping gently. - Use approximately five plates per lysate and 2–5 plates per con-
trol. It is important not to plate too many bacteria on a single
plate; if 100 μL produces a lawn of background growth, reduce
the volume in future experiments to obtain clean colonies. - The approximate concentration of S. aureus SH1000 at
OD 600 = 1 is 2 × 108 CFU/mL. You may need to adjust this
value depending upon your own findings. - The concentration of aliquots from a single strain preparation
should not vary by more than 10%. If they do, your studies
may be affected by imprecise bacterial numbers, and you
should consider remaking the aliquots. The most likely cause
of concentration variation is failure to properly mix the bacte-
rial suspension prior to pipetting. - It is best to perform this plating in duplicate or triplicate so
you can obtain an average value for the final strain ratio (and
dose) used in the infection. - If you intend to homogenize the organs without an automated
machine, place the organs in sterile 7 mL tubes and follow the
rest of these instructions, simply performing the homogeniza-
tion step manually.
Gareth McVicker et al.