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| - | ===Materials===
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| - | * Sterile water with 0.05% tween-80
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| - | * 7H9 medium
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| - | * LB plates with: no marker; 50μg/mL hygromycin and 25μg/mL kanamycin; 25μg/mL kanamycin
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| - | ===Directions===
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| - | # Grow 100mL culture of ''M. smegmatis'' overnight to A<sub>600</sub> = 0.6-0.8.
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| - | # Centrifuge culture at 3,000rpm for 10 minutes at 4°C. Aspirate supernatant and resuspend in same volume of sterile water + 0.05% tween-80. Repeat.
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| - | # Centrifuge once more, and resuspend in 2mL of just sterile water (no tween).
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| - | # Electroporate 400μL plus DNA with 2.5kV, 1kΩ 25μF, and immediately transfer electroporated slurry to 5mL recovery medium.
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| - | # Incubate recovery medium shaking at 37°C for at least 3.5 hours.
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| - | # Centrifuge recovery medium at 3,000 rpm for 10 minutes. Resuspend in 300μL PBS + 0.05% tween-80 and plate out, 100μL per plate, on blank (no antibiotics), kanamycin, and hyg+kan.
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| - | ===Notes===
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| - | * Positive controls: an integrating plasmid (pMV361); no DNA with electroporation on plain LB; no DNA without electroporation on plain LB.
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| - | * Negative controls: no DNA with and without electroporation on antibiotic plates.
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Current revision
