3.3 Genomic DNA
Isolation for
Quantitative PCR
(qPCR) Standards in M.
tuberculosis
1. Culture 10 mL M. tuberculosis (see Subheading 2.1, items
1–3).
2. Use 1 mL of the growing culture to inoculate 2Â universal
tubes with 9 mL 7H9 supplemented with 10% ADC and 0.05%
Tween-80. Incubate at 37
C, 180 rpm until OD 580 1–1.5.
3. Harvest the cells by centrifugation at 2500 Â g for 15 min and
discard the supernatant.
4. Resuspend the pellets in 1 mL breaking buffer (with RNase).
Slowly add a further 19 mL to adjust the final volume of the
bacterial suspension to 20 mL.
5. Centrifuge the cells at 2500 Â g for 15 min and discard the
supernatant.
6. Resuspend the pellets in 0.5 mL breaking buffer and incubate
on ice for 5 min.
7. Transfer the pellet into a tube containing 300 μL pre-washed
glass beads.
8. Bead beat the samples for 15 s at 6.5 m/s.
9. Incubate the tubes on ice for 15 min, centrifuge at 5000 Â g for
1 min, and transfer the lysates into a clean screw-cap
microcentrifuge tube.
10. Wash the beads with 200 μL breaking buffer and combine with
the lysates.
11. Add 1 volume of 10% (w/v) SDS and 10 μL proteinase K.
12. Mix carefully and incubate at 55
C for 3 h, mixing the tubes
occasionally by inverting. The suspension should become
viscous.
13. Steps 11 and 12 (adding SDS and proteinase K and incubating
at 55
C) can be repeated for an additional 1 h to further
remove contaminants.
14. Cool the tubes on ice and add an equal volume of phenol/
chloroform/isoamyl alcohol at 25:24:1.
15. Mix the tubes by inverting 100 times and centrifuge at
12,000 Â g for 30 min.
16. Carefully remove the aqueous layer to a clean screw-cap tube
(see Note 14).
17. Add 0.1 volume of 3 M sodium acetate and 1 volume of
isopropanol and incubate at 4
C for 1 h (alternatively incubate
overnight at À20
C).
18. Centrifuge at 12,000 Â g for 30 min to pellet the
genomic DNA.
19. Remove the supernatant and wash the DNA pellet carefully
with ice-cold 70% (v/v) ethanol.
68
Heena Jagatia and Daire Cantillon
Isolation for
Quantitative PCR
(qPCR) Standards in M.
tuberculosis
1. Culture 10 mL M. tuberculosis (see Subheading 2.1, items
1–3).
2. Use 1 mL of the growing culture to inoculate 2Â universal
tubes with 9 mL 7H9 supplemented with 10% ADC and 0.05%
Tween-80. Incubate at 37
C, 180 rpm until OD 580 1–1.5.
3. Harvest the cells by centrifugation at 2500 Â g for 15 min and
discard the supernatant.
4. Resuspend the pellets in 1 mL breaking buffer (with RNase).
Slowly add a further 19 mL to adjust the final volume of the
bacterial suspension to 20 mL.
5. Centrifuge the cells at 2500 Â g for 15 min and discard the
supernatant.
6. Resuspend the pellets in 0.5 mL breaking buffer and incubate
on ice for 5 min.
7. Transfer the pellet into a tube containing 300 μL pre-washed
glass beads.
8. Bead beat the samples for 15 s at 6.5 m/s.
9. Incubate the tubes on ice for 15 min, centrifuge at 5000 Â g for
1 min, and transfer the lysates into a clean screw-cap
microcentrifuge tube.
10. Wash the beads with 200 μL breaking buffer and combine with
the lysates.
11. Add 1 volume of 10% (w/v) SDS and 10 μL proteinase K.
12. Mix carefully and incubate at 55
C for 3 h, mixing the tubes
occasionally by inverting. The suspension should become
viscous.
13. Steps 11 and 12 (adding SDS and proteinase K and incubating
at 55
C) can be repeated for an additional 1 h to further
remove contaminants.
14. Cool the tubes on ice and add an equal volume of phenol/
chloroform/isoamyl alcohol at 25:24:1.
15. Mix the tubes by inverting 100 times and centrifuge at
12,000 Â g for 30 min.
16. Carefully remove the aqueous layer to a clean screw-cap tube
(see Note 14).
17. Add 0.1 volume of 3 M sodium acetate and 1 volume of
isopropanol and incubate at 4
C for 1 h (alternatively incubate
overnight at À20
C).
18. Centrifuge at 12,000 Â g for 30 min to pellet the
genomic DNA.
19. Remove the supernatant and wash the DNA pellet carefully
with ice-cold 70% (v/v) ethanol.
68
Heena Jagatia and Daire Cantillon