2.2 DNA Isolation
Using ChIP in M.
tuberculosis
1. See Subheading 2.1, items 1–3, for M. tuberculosis liquid culture and Subheading 2.1, items 4 and 5, for solid culture.
2. 40% formaldehyde.
3. 2 M glycine.
4. Phosphate-buffered saline (PBS) pH 7.4 or Tris-buffered saline
(TBS) (20 mM Tris–HCl pH 7.5, 150 mM NaCl).
5. Immunoprecipitation buffer: 50 mM HEPES-KOH pH 7.5,
150 mM NaCl, 1 mM EDTA, 1% (w/v) Triton X-100, 0.1%
(w/v) sodium deoxycholate, 0.1% (w/v) SDS, and antiprotease
mini tablet (1 tablet per 10 mL buffer; Sigma-Aldrich).
6. 0.1 mm silica lysing matrix B beads in 2 mL tubes
(MP Biomedicals).
7. Bioruptor (Diagenode).
8. Protein G or protein A magnetic beads (NEB).
9. Magnetic rack.
10. Nonstick 1.5 mL microcentrifuge tubes (Ambion).
11. IP buffer: 50 mM HEPES-KOH pH 7.5, 150 mM NaCl,
1 mM EDTA, 1% (w/v) Triton X-100, 0.1% sodium deoxycholate, and 0.1% (w/v) SDS.
12. 5Â Elution buffer: TE pH 7.6, 5% (w/v) SDS.
13. IP buffer with salt: 50 mM HEPES-KOH pH 7.5, 500 mM
NaCl, 1 mM EDTA, 1% (w/v) Triton X-100, 0.1% (w/v)
sodium deoxycholate, and 0.1% (w/v) SDS.
14. Wash buffer: 10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA,
0.5% (w/v) Nonidet P-40 substitute, and 0.5% (w/v) sodium
deoxycholate.
15. 5Â Elution buffer: TE pH 7.6, 1% (w/v) SDS.
16. TE buffer pH 8.0.
17. TE buffer pH 7.5.
18. RNase A: 10 mg/mL (Sigma-Aldrich).
19. Proteinase K: 20 mg/mL (Sigma-Aldrich).
20. QIAquick PCR Purification Kit (Qiagen).
21. Tube rotator.
22. Monoclonal or polyclonal antibody raised against protein of
interest.
2.3 Genomic DNA
Isolation for
Quantitative PCR
(qPCR) Standards in M.
tuberculosis
1. M. tuberculosis culture in liquid medium (see Subheading 2.1,
items 1–3).
2. 50 mL Falcon tubes.
3. 25 and 10 mL pipettes.
4. 150–212 μm glass beads.
DNA Isolation from Mycobacteria
63
Using ChIP in M.
tuberculosis
1. See Subheading 2.1, items 1–3, for M. tuberculosis liquid culture and Subheading 2.1, items 4 and 5, for solid culture.
2. 40% formaldehyde.
3. 2 M glycine.
4. Phosphate-buffered saline (PBS) pH 7.4 or Tris-buffered saline
(TBS) (20 mM Tris–HCl pH 7.5, 150 mM NaCl).
5. Immunoprecipitation buffer: 50 mM HEPES-KOH pH 7.5,
150 mM NaCl, 1 mM EDTA, 1% (w/v) Triton X-100, 0.1%
(w/v) sodium deoxycholate, 0.1% (w/v) SDS, and antiprotease
mini tablet (1 tablet per 10 mL buffer; Sigma-Aldrich).
6. 0.1 mm silica lysing matrix B beads in 2 mL tubes
(MP Biomedicals).
7. Bioruptor (Diagenode).
8. Protein G or protein A magnetic beads (NEB).
9. Magnetic rack.
10. Nonstick 1.5 mL microcentrifuge tubes (Ambion).
11. IP buffer: 50 mM HEPES-KOH pH 7.5, 150 mM NaCl,
1 mM EDTA, 1% (w/v) Triton X-100, 0.1% sodium deoxycholate, and 0.1% (w/v) SDS.
12. 5Â Elution buffer: TE pH 7.6, 5% (w/v) SDS.
13. IP buffer with salt: 50 mM HEPES-KOH pH 7.5, 500 mM
NaCl, 1 mM EDTA, 1% (w/v) Triton X-100, 0.1% (w/v)
sodium deoxycholate, and 0.1% (w/v) SDS.
14. Wash buffer: 10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA,
0.5% (w/v) Nonidet P-40 substitute, and 0.5% (w/v) sodium
deoxycholate.
15. 5Â Elution buffer: TE pH 7.6, 1% (w/v) SDS.
16. TE buffer pH 8.0.
17. TE buffer pH 7.5.
18. RNase A: 10 mg/mL (Sigma-Aldrich).
19. Proteinase K: 20 mg/mL (Sigma-Aldrich).
20. QIAquick PCR Purification Kit (Qiagen).
21. Tube rotator.
22. Monoclonal or polyclonal antibody raised against protein of
interest.
2.3 Genomic DNA
Isolation for
Quantitative PCR
(qPCR) Standards in M.
tuberculosis
1. M. tuberculosis culture in liquid medium (see Subheading 2.1,
items 1–3).
2. 50 mL Falcon tubes.
3. 25 and 10 mL pipettes.
4. 150–212 μm glass beads.
DNA Isolation from Mycobacteria
63