Specific strains transformable with SA08B-conditioned plasmids include laboratory strain Newman, laboratory strain
ATCC29213, laboratory strain N315, USA300 isolates,
CC239/ST239 isolates, and CC5/ST5 isolates [11, 24].
3. SA30B. SA30B is a derivative of DC10B which actively mimics
the methylation profile of S. aureus CC30/ST30 [24]. Use of
this strain conditions plasmids for introduction into S. aureus
laboratory strain Cowan and MRSA252, among other CC30
and CC30-restriction compatible strains [24].
2.3 Materials
for Electrocompetent
Cell Preparation
1. B2 broth [25] (1.0% casein hydrolysate, 2.5% yeast extract,
0.1% K 2 HPO 4 , 0.5% glucose, 2.5% NaCI, pH 7.5), LB broth
(Fisher Scientific), or TSB broth (Becton Dickinson) as
appropriate.
2. Sterile 10% glycerol (autoclaved), chilled on ice.
3. Sterile distilled water (autoclaved), chilled on ice.
2.4 Electroporation
Materials
1. Pellet Paint
® NF Co-Precipitant (Millipore).
2. Ethanol (100% and 70%).
3. TE buffer.
4. Sterile sucrose 1.5 M (filter sterilized).
5. Sterile 10% glycerol (autoclaved).
6. 0.1 cm electrode gap electroporation cuvettes
7. TSB broth or SOC broth (NEB), as appropriate.
8. Electroporator (Bio-Rad MicroPulser, or equivalent).
9. LB plates with appropriate selective antibiotics (ampicillin
100 μg/mL, chloramphenicol 10 μg/mL, or chloramphenicol
10 μg/mL with erythromycin 10 μg/mL).
2.5 Materials
for Counterselection
Vector Preparation
1. BsaI-HF (NEB).
2. CIP (NEB).
3. T4 PNK (NEB).
4. T4 Ligase buffer (NEB).
5. T4 DNA Ligase (NEB).
6. Primers sgRNA_check_F (5
0 -AAAAATATGAACACTCTAT
CATTG-3
0 ) and sgRNA_check_R (5
0 -CGGTGCCACTTTTT
CAAGTT-3
0 ).
7. KAPA HiFi 2Â Hotstart Readymix (KAPA Biosystems) or
equivalent.
2.6 Antibiotics
Ampicillin (stock at 100 mg/mL, prepared in laboratory grade
water).
Chloramphenicol (stock at 34 mg/mL, prepared in 100% Ethanol).
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Kelsi Penewit and Stephen J. Salipante
ATCC29213, laboratory strain N315, USA300 isolates,
CC239/ST239 isolates, and CC5/ST5 isolates [11, 24].
3. SA30B. SA30B is a derivative of DC10B which actively mimics
the methylation profile of S. aureus CC30/ST30 [24]. Use of
this strain conditions plasmids for introduction into S. aureus
laboratory strain Cowan and MRSA252, among other CC30
and CC30-restriction compatible strains [24].
2.3 Materials
for Electrocompetent
Cell Preparation
1. B2 broth [25] (1.0% casein hydrolysate, 2.5% yeast extract,
0.1% K 2 HPO 4 , 0.5% glucose, 2.5% NaCI, pH 7.5), LB broth
(Fisher Scientific), or TSB broth (Becton Dickinson) as
appropriate.
2. Sterile 10% glycerol (autoclaved), chilled on ice.
3. Sterile distilled water (autoclaved), chilled on ice.
2.4 Electroporation
Materials
1. Pellet Paint
® NF Co-Precipitant (Millipore).
2. Ethanol (100% and 70%).
3. TE buffer.
4. Sterile sucrose 1.5 M (filter sterilized).
5. Sterile 10% glycerol (autoclaved).
6. 0.1 cm electrode gap electroporation cuvettes
7. TSB broth or SOC broth (NEB), as appropriate.
8. Electroporator (Bio-Rad MicroPulser, or equivalent).
9. LB plates with appropriate selective antibiotics (ampicillin
100 μg/mL, chloramphenicol 10 μg/mL, or chloramphenicol
10 μg/mL with erythromycin 10 μg/mL).
2.5 Materials
for Counterselection
Vector Preparation
1. BsaI-HF (NEB).
2. CIP (NEB).
3. T4 PNK (NEB).
4. T4 Ligase buffer (NEB).
5. T4 DNA Ligase (NEB).
6. Primers sgRNA_check_F (5
0 -AAAAATATGAACACTCTAT
CATTG-3
0 ) and sgRNA_check_R (5
0 -CGGTGCCACTTTTT
CAAGTT-3
0 ).
7. KAPA HiFi 2Â Hotstart Readymix (KAPA Biosystems) or
equivalent.
2.6 Antibiotics
Ampicillin (stock at 100 mg/mL, prepared in laboratory grade
water).
Chloramphenicol (stock at 34 mg/mL, prepared in 100% Ethanol).
130
Kelsi Penewit and Stephen J. Salipante