14. Chloroform–isoamyl alcohol (24:1) solution: Add 2 mL of
molecular-grade isoamyl alcohol to 48 mL molecular-grade
chloroform and mix by inversion ten times. Assign a shelf life
of 3 days if stored at ambient temperature.
15. 100% molecular-grade isopropanol.
16. 70% molecular-grade ethanol.
3 Methods
3.1 Isolation of DNA
from M. tuberculosis
for Detection of
Plasmid Inserts
1. Pick a colony of M. tuberculosis containing the electroporated
plasmid from 7H11 solid medium supplemented with the
antibiotic for isolation of cells containing the desired plasmid,
and use this to inoculate 5 mL 7H9 supplemented with 10%
ADC, 0.05% Tween-80, and antibiotic until OD 600 0.6–0.8
(see Notes 1 and 2).
2. Heat-kill 1 mL of the culture by placing in a water bath at 98
C
for 2 h (see Note 3).
3. Centrifuge at 13,000 Â g for 10 min.
4. Resuspend the pellet in 100 μL nuclease-free water.
5. Add equal volume of 100% chloroform and vortex vigorously.
6. Centrifuge at full speed for 1 min.
7. Carefully, pipette the upper phase into a clean 1.5 mL microcentrifuge tube, this contains the genomic DNA, which is ready
to use for PCR.
3.2 DNA Isolation
Using ChIP in M.
tuberculosis
1. Culture the M. tuberculosis in 5 mL 7H9 supplemented with
10% ADC and 0.05% Tween-80. Incubate at 37
C, 180 rpm
for 6–7 days (see Note 4).
2. Inoculate 70 mL 7H9, supplemented with ADC with 1 mL of
M. tuberculosis preculture, and incubate to OD 600 0.4–0.6 at
37
C (see Subheading 2.1, items 1–3).
3. Add 1% formaldehyde to the 70 mL cultures and incubate for
20 min at 37
C.
4. Plate a small sample onto 7H11 solid agar to confirm the
sterilization of the M. tuberculosis cultures and incubate at
37
C for 4–6 weeks.
5. Add 0.05 M glycine to the cultures and incubate for 5 min to
quench the cross-linking.
6. Harvest the cells by centrifugation at 4427 Â g force for 5 min
at 4
C.
7. Wash the cells twice in 25 mL PBS or TBS.
8. Store the pellets at À80
C (see Note 5).
DNA Isolation from Mycobacteria
65