9. 7H10-KHGm plates (see Note 1): Dissolve 19 g of Middlebrook 7H10 medium (Difco) in 900 mL deionized water. Autoclave. When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add kanamycin stock to give final concentration of
20μg/mL, hygromycin to give final concentration of 50μg/
mL, and gentamicin to give a final concentration of 10μg/mL.
Mix well and pour in standard plastic Petri dishes.
10. Complementation plasmid (up to 1μg in a volume not exceeding 5μL), as described in Subheading 3.1.
11. Electroporation cuvettes with a 2 mm gap.
12. Electroporation equipment capable of producing a pulse of
2.5 kV, 1000 Ω, 25μF.
13. Sucrose: make a 50% (w/v) stock solution in distilled water.
Filter sterilize. Use at a final concentration of 2% (w/v) for
M. tuberculosis.
14. 7H10-GmXSuc plates (see Note 1): Dissolve 19 g of Middlebrook 7H10 medium (Difco) in 900 mL deionized water. Autoclave. When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add gentamicin to give a final concentration of
10μg/mL, X-gal to give a final concentration of 50μg/mL,
and sucrose to give a final concentration of 2%. Mix well and
pour in standard plastic Petri dishes.
15. 1 mL (vol) 1 mm sterile glass beads.
16. Primers designed to overlap the deletion region.
17. Heat block set to 100
C.
18. 10 mM Tris pH 8.0.
19. 0.2μM 15 mm filter units.
20. 3 mL LuerLok syringes.
21. PCR reagents, thermocycler, and consumables.
22. PCR cleanup or gel extraction Kit, e.g., QiaQuick (Qiagen) or
similar.
23. Agarose gel electrophoresis equipment.
24. Southern blotting equipment.
2.3 Confirming
Essentiality by Gene
Switching
1. Delinquent (Δ
0 int) strain from Subheading 3.2.
2. pUC-Hyg-Int or pHAPY (available from Tanya Parish, Tanya.
Parish@seattlechildrens.org).
3. 490 cm
3 roller bottles (Corning).
4. Roller incubator (set at 37
C).
5. 2 M glycine. Filter sterilize. Store at 4
C.
6. Oleic albumin dextrose catalase (OADC) supplement (Becton
Dickinson). Store at 4
C and use at a final concentration of
10% v/v.
292
Amanda Claire Brown
C, add 100 mL of OADC
enrichment. Add kanamycin stock to give final concentration of
20μg/mL, hygromycin to give final concentration of 50μg/
mL, and gentamicin to give a final concentration of 10μg/mL.
Mix well and pour in standard plastic Petri dishes.
10. Complementation plasmid (up to 1μg in a volume not exceeding 5μL), as described in Subheading 3.1.
11. Electroporation cuvettes with a 2 mm gap.
12. Electroporation equipment capable of producing a pulse of
2.5 kV, 1000 Ω, 25μF.
13. Sucrose: make a 50% (w/v) stock solution in distilled water.
Filter sterilize. Use at a final concentration of 2% (w/v) for
M. tuberculosis.
14. 7H10-GmXSuc plates (see Note 1): Dissolve 19 g of Middlebrook 7H10 medium (Difco) in 900 mL deionized water. Autoclave. When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add gentamicin to give a final concentration of
10μg/mL, X-gal to give a final concentration of 50μg/mL,
and sucrose to give a final concentration of 2%. Mix well and
pour in standard plastic Petri dishes.
15. 1 mL (vol) 1 mm sterile glass beads.
16. Primers designed to overlap the deletion region.
17. Heat block set to 100
C.
18. 10 mM Tris pH 8.0.
19. 0.2μM 15 mm filter units.
20. 3 mL LuerLok syringes.
21. PCR reagents, thermocycler, and consumables.
22. PCR cleanup or gel extraction Kit, e.g., QiaQuick (Qiagen) or
similar.
23. Agarose gel electrophoresis equipment.
24. Southern blotting equipment.
2.3 Confirming
Essentiality by Gene
Switching
1. Delinquent (Δ
0 int) strain from Subheading 3.2.
2. pUC-Hyg-Int or pHAPY (available from Tanya Parish, Tanya.
Parish@seattlechildrens.org).
3. 490 cm
3 roller bottles (Corning).
4. Roller incubator (set at 37
C).
5. 2 M glycine. Filter sterilize. Store at 4
C.
6. Oleic albumin dextrose catalase (OADC) supplement (Becton
Dickinson). Store at 4
C and use at a final concentration of
10% v/v.
292
Amanda Claire Brown